Immunoproteomic Approach to Elucidating the Pathogenesis of Cryptococcosis Caused by Cryptococcus gattii

Sarah E. Jobbins, Cameron J. Hill, Jocelyn M. DSouza-Basseal, Matthew P. Padula, Ben R. Herbert, and Mark B. Krockenberger*,
The Faculty of Veterinary Science and the School of Molecular and Microbial Biosciences, the University of Sydney, and the Proteomics Technology Centre of Expertise, Faculty of Science, University of Technology Sydney, Australia
Received January 14, 2010

Cryptococcosis caused by Cryptococcus gattii is a devastating disease of immunocompetent hosts with an incompletely understood pathogenesis. Utilizing an immunoproteomic approach in a naturally occurring koala model of disease, a number of key proteins and pathways are identied in the early and late pathogenesis of cryptococcosis for the rst time. In particular, the thioredoxin system appears important in the pathogenesis of cryptococcosis caused by C. gattii VGII.
Keywords: Cryptococcus gattii cryptococcosis subclinical cryptococcosis koala Phascolarctos cinereus immunoproteomics

Cryptococcosis is a life-threatening systemic fungal disease of humans and animals, manifested predominantly by respiratory tract disease and meningoencephalitis.1 It is caused by the Cryptococcus species complex,2 which includes C. neoformans and C. gattii. While C. neoformans is a ubiquitous environmental organism found worldwide in bird guano3 and soil4 and an important cause of disease in immunocompromised individuals,1 C. gattii has a more restricted environmental distribution and typically affects apparently healthy, immunocompetent hosts.1 There are four genetically distinct molecular types of C. gattii (VGI to VGIV),57 of which VGI and VGII are the most important in Australia. VGI is the most common molecular type isolated from clinical and environmental samples (predominantly eucalypts8,9) in Australia10,11 and worldwide.5,6 VGII isolates have less well-dened environmental associations and account for regionally high incidences of cryptococcosis, particularly in Western Australia,12,13 Arnhem Land in the Northern Territory10 of Australia (regional incidence of up to 140 cases/ million people/year14), and on Vancouver Island, British Columbia, Canada15,16 (regional incidence 36 cases/million people/ year17 since 1999). Disease is the outcome of interactions among the host, the pathogen, and the environment. VGII appears to be signicantly better at sexual reproduction in the environment and producing infectious propagules than VGI,18 which may be the reason for the increased incidence and outbreak epidemiology of cryptococcosis in areas where VGII predominates. Alternatively, this could be a result of other virulence factors.
* To whom correspondence should be addressed. Mark B. Krockenberger, Faculty of Veterinary Science, The University of Sydney, Australia, 2006. Tel: +61 2 9351 2023. Fax: +61 2 9351 7421. Email: mark.krockenberger@sydney. edu.au. Faculty of Veterinary Science. Proteomics Technology Centre of Expertise. School of Molecular and Microbial Biosciences.

While numerous pathogen virulence factors have been dened, their relative importance in pathogenesis is largely unknown and their interaction with the host response is unclear. Primary infection occurs via inhalation of the infectious propagule19 into the respiratory tract. In some cases, fungi gain access to the bloodstream, through which they disseminate and can affect multiple organ systems, with particular tropism for the CNS.1 The ubiquity of the fungus in the environment and the comparatively rare occurrence of clinical disease are suggestive that subclinical disease20,21 could be common and that the host response is adequate to contain or eliminate the pathogen in the vast majority of instances in which contact occurs. Cryptococcosis in the koala (Phascolarctos cinereus) is a model of naturally occurring disease2225 that can be exploited to investigate the pathogenesis of cryptococcosis caused by C. gattii. The koala shares a known ecological niche of C. gattii, eucalypts, and is therefore exposed to the organism regularly. Close to 100% of koalas demonstrate colonization of the nasal mucosa22 and up to 50% in some captive populations have subclinical cryptococcal disease.24 Clinical cryptococcosis is of much lower prevalence, affecting approximately 3-5% of koalas (Krockenberger, M.B. Pers. comm.), and usually presents initially as respiratory tract disease (upper and lower) before dissemination to the CNS and other tissues.25 The vast majority of koala cryptococcosis is caused by C. gattii and the proportions affecting the CNS, lower respiratory tract,and skin are comparable with ndings in humans,25 highlighting the utility of the koala as a naturally occurring model for the pathogenesis of human cryptococcosis. The advantages of a naturally occurring model of cryptococcosis, including inoculation, breach of host defenses, and early pathogenesis, can be utilized to complement information gleaned from laboratory animal investigations that utilize large inocula and compromise of host defenses to initiate disease.
10.1021/pr100028t 2010 American Chemical Society

3832 Journal of Proteome Research 2010, 9, 38323841

Published on Web 06/14/2010

Immunoproteomics of Koala Cryptococcosis

Table 1. Details of the 6 Koalas Used in This Study
ID animal status infective isolatea LCAT titer sex

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C1 C2 SC1 SC2 B1 B2
a

Rambo Neo Angel Clancy Vic 1 Vic 2

Clinical Clinical Subclinical Subclinical Control/Background Control/Background

C. gattii, VGI C. gattii, VGII -

64 32 768 4 2 0 0

M M F M NR NR

Where infective isolate was able to be determined; NR not recorded.

Immunoproteomics uses circulating antibodies from an affected host to detect disease-associated antigens in pathogen protein macroarrays and has been successfully used to identify biomarkers for many diseases including systemic candidiasis26 and malaria.27 Immunoproteomics selectively identies pathogen proteins that interact with the host immune system, thus reducing the necessity to examine the full proteome. Successful utilization of this technique requires an effective protein isolation technique and is sensitive to nonprotein contaminants (e.g., cell wall debris and polysaccharides) in the protein preparation,28 of particular relevance to C. gattii which has an extremely large polysaccharide capsule. Most previous studies of C. neoformans have focused on secreted proteins in culture ltrates (reviewed in ref 29) or cell surface associated proteins,30 potentially to avoid the problem of the capsule. In this study, we demonstrate a classic immunoproteomic strategy to investigate the C. gattii immunome, using the naturally occurring koala model of cryptococcosis. The large capsule of C. gattii makes cell lysis difcult, and many proteins can be lost due to nonspecic binding to capsular polysaccharides. To enable comprehensive proteomics, we developed a novel protocol for the efcient extraction of cryptococcal proteins for use in 2-dimensional gel electrophoresis (2-DE), which minimizes interference due to capsular polysaccharide in cell preparations. The disease-associated antigens identied could form future targets for therapy or diagnostic biomarkers and indicators of prognosis.

Figure 1. Representative 2-DE prole of Cryptococcus gattii, demonstrating protein distribution with bias toward low molecular weight proteins.

Materials and Methods

Fungal Isolates and Culture Conditions. R265 (C. gattii molecular type VGII) was used in the development of the protein extraction protocol and 571000 (C. neoformans), 571071 (C. gattii molecular type VGI) and MK992f (C. gattii molecular type VGII) were used for immunoproteomic analyses (Table S1, Supporting Information). R265 was supplied by Jocelyn DSouza-Basseal. The remaining isolates were obtained from the Veterinary Pathobiology Freeze-Dried Collection, Faculty of Veterinary Science at the University of Sydney, Australia. The isolates were subcultured in 50 mL Sabourauds Dextrose Broth (SDB) at 33 C for 5 days. C. gattii isolates from disease cases were identied to molecular type using a modied version of the URA5 gene restriction digest.6,31 Koala Cryptococcosis Patients. Serum specimens from four koala patients with cryptococcosis (2 koalas with subclinical cryptococcosis and 2 with clinical cryptococcosis) were obtained during routine screening of captive animals, or as part of ongoing monitoring of clinical cases (Table 1). Diagnosis of subclinical disease was made after detection of circulating capsular antigen without any clinically observable signs of disease.24 This group of animals typically has a microscopic focus of tissue invasion.24,25 Clinical cryptococcosis

was diagnosed when clinically evident signs of cryptococcal disease were accompanied by the detection of capsule in circulation and the identication of a focus of disease by culture and histopathology.24,25 Due to the nature of naturally occurring cryptococcosis, the exact time-course since exposure to the organism, prior to sampling, was not available. Sera from 2 healthy koalas with no history of cryptococcosis and no detectable antigen titer, from an area of low environmental presence of C. gattii, were used as controls to determine background reactivity due to cross-reactivity with similar epitopes on other organisms and/or low grade environmental exposure (Table 1). Development of an Efcient Protocol for Extraction of Cryptococcal Proteins. In our initial protocol, cultured Cryptococcus cells were washed three times in 15 mL of PBS (pH 7.4) containing a protease inhibitor cocktail (Roche Applied Science, Australia), and the resulting cell pellet was suspended in extraction buffer (1% (w/v) C7bz0, 2 M Thiourea, 7 M Urea, 40 mM Tris). The sample was then disrupted in a beadmill at maximum intensity for 5 min, followed by four 30 s bursts of 70% power ultrasonication, with the sample placed on ice for 30 s between bursts. The disrupted cells were then reduced and alkylated with 10 mM Tributylphosphine and 10 mM Acrylamide at ambient temperature for 1 h.32 Cell debris was pelleted by centrifugation at 16 000 g for 10 min and the pellet discarded. The proteins in the supernatant were precipitated with 5 volumes of acetone for 30 min at ambient temperature and collected by centrifugation at 4000 g for 10 min. The protein pellet was then solubilized in either LDS Sample Buffer (Invitrogen, Australia) for one-dimensional electrophoresis (1-DE), or extraction buffer (without Tris) for two-dimensional electrophoresis (2-DE). Preliminary experiments using this protocol produced 2-DE proles with protein distributions which were heavily biased toward low molecular weight proteins (Figure 1). To dissociate proteins from capsular material and improve recovery, we investigated the inclusion of cations (LiCl, NaCl or CaCl2) at three concentrations (50 mM, 100 mM and 200 mM) in the extraction buffer. Compared to the other cations, lithium chloride provided optimal protein recovery at 50 mM and was included in the extraction buffer during cell disruption in subsequent experiments. The 2-DE prole produced using
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Figure 2. 2-DE prole produced using 50 mM LiCl in the extraction buffer, demonstrating an improvement in the recovery of high molecular weight proteins. Note the artifactual streaking in the acidic portion of the gel.

this revised protocol showed an improvement in high molecular weight protein recovery; however, it also demonstrated signicant artifactual streaking in the acidic portion of the gel (Figure 2), suggestive of cell wall/capsular material contamination of the rst dimension IEF. To remove potential capsular and cell wall contaminants, we ltered the samples in 100 or 300 kDa centrifugal ltration devices in the presence of 50 mM LiCl and then buffer exchanged them into 1% (w/v) C7bz0, 2 M thiourea, 7 M urea in Micro Bio-Spin 6 Chromatography columns (Bio-Rad, Australia) to remove residual LiCl prior to focusing. Filtration of the protein preparation through a 100 kDa lter successfully eliminated the majority of artifacts in the acidic portion of the 2-DE gel (Figure 3). Therefore, the nal extraction protocol used to create 2-DE protein macroarrays for immunoblotting involved ltration of extracts through a 100 kDa lter and the inclusion of 50 mM LiCl in the extraction buffer and during ltration. 2-DE. Four-hundred micrograms of whole cell protein extract was separated in the rst dimension on an IPG strip (11 cm, pH 4-7 or pH 3-10, Bio-Rad, Australia). The focused IPG strip was equilibrated in 2% (w/v) SDS, 6 M Urea, 250 mM Tris-HCl (pH 8.5) and then separated in the second dimension on a 4-12% Bis-Tris gel at 160 V. Following separation, gels were visualized by staining with either Flamingo Fluorescent Gel Stain (Bio-Rad, Australia) or Coomassie Blue G-250 or alternativelytransferredtonitrocellulosemembranesforimmunoblotting. Immunoblot Analysis. Separated cryptococcal proteins were transferred onto 0.45 m nitrocellulose membranes by the semidry method of Khyse-Anderson,33 with modications as follows. The blotting stack was initially formed with an additional membrane on the cathode side of the protein gel. Following 5 min of protein transfer in the reverse direction at 300 mA, the cathode membrane was removed and stained for 2 h with Direct Blue 71 (DB71). The blotting stack was then reassembled with the polarity now in the traditional orientation, and the proteins were transferred onto the anode membrane at 300 mA for 30 min. The anode membrane was then incubated with 5% nonfat milk powder in PBS containing 0.1% (v/v) Tween-20 (PBS-T) for 1 h at 4 C, in preparation for hybridization with sera. The blocked membrane was washed with PBS-T 3 times, for 10 min each wash, and incubated with patient sera (1:10 dilution) overnight at 4 C. Following another
3834 Journal of Proteome Research Vol. 9, No. 8, 2010

Figure 3. 2-DE proles produced when samples were ltered in the presence of 50 mM LiCl through (A) a 300 kDa centrifugal lter and (B) a 100 kDa centrifugal lter. Filtration of the protein preparation through a 100 kDa lter successfully eliminated the majority of artifacts in the acidic portion of the 2D gel.

three washes in PBS-T, the membrane was incubated with rabbit antiopossum IgG (heavy and light chain) antibody (Bethyl Laboratories Inc., Montgomery, TX, 1:1000 dilution) for 1.5 h at ambient temperature, and then washed three times as described above. The membrane was then incubated with an alkaline phosphatase-conjugated antirabbit IgG (Chemicon International, Billerica, MA, 1:1000 dilution) for 1.5 h at ambient temperature, and washed three times in TBS containing 0.1% (v/v) Tween-20 (TBS-T). Colorimetric development of the immunoblot was achieved using an Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad, Australia). Protein Identication. The stained 2-DE gels, immunoblots, and DB71 stained cathode membrane were scanned on a atbed scanner and aligned using PDQuest software (Bio-Rad, Australia) to enable correlation of the gel image with the DB71 membrane and, subsequently, the DB71 membrane with the immunoblot. Protein spots of interest were excised manually from Coomassie Blue-stained gels and processed for mass spectrometry. Briey, the gel pieces were destained with 50% (w/v) 50 mM NH4HCO3 in 50% acetonitrile (ACN) and dried by evaporation, before being digested in 12.5 ng/L Trypsin in NH4HCO3 overnight at 37 C. The peptides were then extracted from the gel piece with 50% ACN/2% Formic Acid (FA) and the sample concentrated to 15 L in a vacuum centrifuge, prior to analysis with the Tempo nanoLC System (Eksigent, Dublin, CA). Ten microliters of the sample was loaded at 20 L/min with 2% ACN, 0.2% FA (solvent A) onto a C18 reversed phase trap column connected to a 10-way switching valve. After washing the trap for two minutes, the 10-way valve was

Immunoproteomics of Koala Cryptococcosis

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Figure 4. Screening for anticryptococcal antibodies in sera from koalas with cryptococcosis. (A) Coomassie blue staining of C. gattii proteins separated by 2-DE. (B) Representative result of immunoblot with sera from a control koala. (C) Representative result of immunoblot with serum from a koala with subclinical disease. (D) Representative result of immunoblot with serum from a koala with clinical disease. Numbered spots indicate reactive proteins, the details of which can be found in Table 2.

switched and the peptides were washed off the trap at 300 nL/ min onto a New Objective IntegraFrit column (75 M ID 100 mm) packed with ProteoPep II C18 resin. At the moment of switching, a gradient program was started to elute bound peptides from the column and into the source of a QSTAR Elite hybrid quadrupole-time-of-ight mass spectrometer (Applied Biosystems/MDS Sciex). This program consisted of the following phases: 5-30% solvent B (98% ACN + 0.2% FA) over 8 min, 30-80% solvent B over 2 min, 80% solvent B for 2 min, 80-5% solvent B for 3 min, re-equilibrate for 10 min in solvent A prior to the next sample. Eluted peptides owed from the column into a MicroIonSpray II-mounted 75 M ID emitter tip that tapered to 15 M. Charged peptides were then ionised by nanoelectrospray with 2300 V into the source of the QSTAR which then performed an Intelligent Data Acquisition (IDA) experiment. Briey, a mass range of 375-1500 Da was continuously scanned until a peptide of charge state 2+ to 5+ was detected with an intensity of more than 100 counts/second. This precursor peptide was then selected, fragmented and the product ion fragment masses measured over a mass range of 100-1500 Da. The mass of the precursor peptide was then excluded for 45 s, so that lower abundance peptides would also be fragmented and analyzed. The MS/MS data les produced by the QSTAR were then used to search LudwigNR using the Mascot algorithm (version 2.1.0, provided by the Australian Proteomics Computational Facility, http://www.apcf.edu.au/34) with parameter settings as follows. Fixed modications: propionamide. Variable modications: oxidized Methionine. Enzyme: Trypsin. Number of allowed missed cleavages: 3. Peptide mass tolerance: 100 ppm. MS/MS mass tolerance: 0.2 Da. Charge state: 2+ and 3+.

Results and Discussion

The development of a successful whole-cell protein extraction and purication protocol from C. gattii facilitated the use

of 2-DE and immunoblotting to identify important proteins in the pathogenesis of cryptococcosis, several of which are potential candidates for diagnostic and therapeutic applications. Previous work has focused mainly on cryptococcal culture ltrate preparations (CneF)35,36 and/or cell surface associated proteins.30 The use of proteins extracted from the entire yeast cell allows an assessment of a larger proportion of the cryptococcal proteome, more reective of natural disease, where both internal and external proteins are exposed to the immune system. The key step in extracting the cryptococcal proteome was removal of the thick polysaccharide capsule (especially large in C. gattii). Standard extraction protocols resulted in loss of high molecular weight proteins (in comparison to C. neoformans) as well as streaking in the acidic portion of the gels. Bacterial cell wall material has been shown to bind proteins in 2-DE preparations,28 and the highly viscous nature of the cryptococcal capsule may have similarly led to entrapment of high molecular weight proteins. Capsular carbohydrates, including glucuronoxylomannan fragments (GXM, 1.2-1.5 106 Da) and galactoxylomannan (GalXM, 1 105 Da), are small enough to enter IPG strips, disrupting the subsequent 2-DE gel.28 In the methodology presented here, cations were utilized to effectively disrupt the capsule and dissociate proteins from capsular material, resulting in a large increase in both high molecular weight proteins and total protein recovered. The combination of LiCl and ltration to remove capsular fragments prevented streaking in the acidic portion of the gels. Immunoblot analysis (Figure 4) of the diseased cohort of animals detected a total of 53 distinct protein spots in the three Cryptococcus isolates examined. Successful identications for 48 out of 53 spots were obtained by LC-MS/MS and corresponded to 37 unique proteins (Table 2, Figure 5). Of these, 6 proteins (ATP synthase subunit beta, UDP-glucose 6-dehydrogenase, phosphoglycerate
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Table 2. Immunodominant Proteins Identied by Serologic Proteome Analysis

functiona locationa

3836 Oxidative phosphorylation. Mitochondrion inner membrane. Cell cycle regulation. Immunogen in mouse model of cryptococcosis.30 Regulation of mitotic cell cycle. Ribosomal protein. Cytoplasm and nucleus. Component of C. neoformans virulence bags.59 Cytosol and nucleus. Cytoplasm. Component of C. neoformans virulence bags.59 C. neoformans virulence, viability.38 Mitochondrion and cytoplasm.38 Component of C. neoformans virulence bags.59 Cytoplasm. Component of C. neoformans virulence bags.59 Cytoplasm. Component of C. neoformans virulence bags.59 Cytoplasm. C. neoformans virulence, defense against oxidative stress, survival inside macrophages.37 Defense against oxidative stress, C. neoformans virulence, growth at 25 and 38.5 C.41 Upregulated in C. neoformans during growth at 37 C and oxidative stress.41 Chaperone, HSP70 family member. HSP70 is an immunogen in mouse model of cryptococcosis.30 Chaperone, HSP60 family member. Cytoplasm. Component of C. neoformans virulence bags.59 Cytoplasm. Cytoplasm. Component of C. neoformans virulence bags.59 Cytosol and cell wall.52 Component of C. neoformans virulence bags.59 Cytoplasm and nucleus. Mitochondrion. Plasma membrane. Chaperone, HSP70 family member. HSP70 is an immunogen in mouse model of cryptococcosis.30 Tissue invasion in C. albicans,53 stimulates protective IgG2 in systemic candidiasis.65 Immunogen in mouse model of cryptococcosis.30 Post-translational protein modication. Protein synthesis. Glycolytic enzyme. Plasminogen-binding protein in C. neoformans,52 stimulates protective IgG2 in systemic candidiasis.66 Pyruvate metabolic process, transferase activity. Oxidative phosphorylation. Pyruvate dehydrogenase complex. Mitochondrion inner membrane. Cytoplasm and cytoskeleton. Component of C. neoformans virulence bags.59 Cytoplasm and cytoskeleton. Unknown. Unknown. Cytoskeleton protein; depolymerisation stimulates phagosomal extrusion of Cryptococcus from macrophages.67 Actin cytoskeleton organization. Transferase activity. Unknown function. Cytoplasm.

no

ID, accession no

Proteins identied in early pathogenesis (subclinical disease only) 26 NADH-ubiquinone oxidoreductase 30.4 kDa subunit, putative (C. neoformans), Q5KF23 28 14-3-3 protein, putative (C. neoformans), Q5K8Z6

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29 33

Wos2 protein (P21), putative (C. neoformans), Q5K9R0 40S ribosomal protein S0, putative, Q5KAZ6

Proteins identied in late pathogenesis (clinical disease only) 2 Thioredoxin reductase (C. neoformans), Q5KFM3

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10

11

Thioredoxin (allergen cop c2), putative (C. neoformans), Q5KK55 Thiol-specic antioxidant protein 1 (C. neoformans var. grubii), Q7Z9J6 Thiol-specic antioxidant protein 3 (C. neoformans var. grubii), Q7Z9J5 Heat shock protein sks2, putative (C. neoformans), Q5KPD7 Heat shock protein, putative (C. neoformans), Q5KLW7 Heat shock protein, putative (C. neoformans), Q5K8W5 Enolase (C. neoformans), Q5KLA7

14 15

18

Ubiquitin carrier protein (C. neoformans), Q5KF72 Elongation factor ts (Ef-ts), putative (C. neoformans), Q5KKE5 Pyruvate kinase (C. neoformans), Q5KKG6

22

27

30

Dihydrolipoyllysine-residue acetyltransferase, putative (C. neoformans), Q5KIM3 Ubiquinol-cytochrome C reductase complex core protein 2, putative (C. neoformans), Q5K8U4 Actin (C. neoformans), Q5KP06

31 35 37

Prolin (C. neoformans), Q5KNQ6 Transferase, putative Q5KHG0 Expressed protein (putative, uncharacterised protein) Q5KPX0

Jobbins et al.

Proteins identied in both early and late pathogenesis (subclinical and clinical disease) 4 Thioredoxin peroxidase, putative (C. neoformans), Defense against oxidative and nitrosative stress, Q5K9Z8 survival inside macrophages, C. neoformans virulence in mice.41

Table 2 Continued
functiona locationa

no

ID, accession no

Heat shock protein, putative (C. neoformans), Q5K7L0

12 Amino acid metabolism. Cytosol. Cytosol. Mitochondrion.

Chaperone, HSP70 family member. HSP70 is an immunogen in mouse model of cryptococcosis.30 Amino acid metabolism.

13

Cytoplasm. Component of C. neoformans virulence bags.59 Mitochondrion. Component of C. neoformans virulence bags.59 Mitochondrion.

20

Acetohydroxy acid reductoisomerase (C. neoformans), Q96VZ5 Ketol-acid reductoisomerase, putative (C. neoformans), Q5KFA0 Triosephosphate isomerize (C. neoformans), Q5KG36 Glycolytic enzyme; stimulates protective IgG2 in systemic candidiasis.66 Pentose phosphate pathway. Citric Acid Cycle.

Immunoproteomics of Koala Cryptococcosis

21

23

24

Ribose-5-phosphate iosomerase (C. neoformans), Q5KHW6 Succinate-CoA ligase (ADP forming), putative (C. neoformans), Q5KN95 ATP synthase subunit alpha (C. neoformans), Q5KFB9 ATP synthesis. Plasminogen-binding protein in C. neoformans.52 Cytoskeleton protein. Unknown function.

32

Alpha tubulin, putative (C. neoformans), Q5KM62

36

Expressed protein (putative, uncharacterised protein) Q5KMG7

Mitochondrion inner membrane. Component of C. neoformans virulence bags.59 Cytoskeleton. Component of C. neoformans virulence bags.59 Unknown.

Component of C. neoformans virulence bags.59 Eukaryotic translation elongation factor 1 complex. Mitochondrion. Component of C. neoformans virulence bags.59 Cell surface. Mitochondrion inner membrane. Component of C. neoformans virulence bags.59 Cytoplasm.

Proteins identied in Background cohort and excluded from further analyses 1 UDP-glucose 6-dehydrogenase (C. neoformans), C. neoformans virulence, capsule biosynthesis, growth Q5K899 at 37 C, cell wall integrity.68 16 Elongation factor 1-beta (Ef-1-beta), putative (C. Protein synthesis. neoformans), Q5KKD1 17 Pyruvate dehydrogenase e1 component beta subunit, Oxidative decarboxylation. Adhesin in Mycoplasma mitochondrial (C. neoformans), Q5KI1 pneumoniae.69 19 Phosphoglycerate kinase (C. neoformans), Q5KE00 Glycolytic enzyme. Plasminogen-binding protein in C. neoformans.52 25 ATP synthase subunit beta (C. neoformans), Q5KFU0 ATP synthesis. Plasminogen-binding protein in C. neoformans.52 34 Mannose-1-phosphate guanyltransferase (C. Cell wall biosynthesis. neoformans), Q5KKH2

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a Biological functions and locations are referenced when an association with C. neoformans or other fungal pathogens has been described. General functions were obtained from UniProt (www.uniprot.org/) on the basis of their role in other models.

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of both subclinical and clinical groups. A member of the heat shock protein (HSP) 60 family was the only protein identied in all three isolates examined. Alpha-tubulin, succinate-CoA ligase, thioredoxin, prolin and a putative transferase were identied in C. gattii, but not in C. neoformans. Conversely, an HSP70 family member, tsap3 and an uncharacterised protein were only identied in the C. neoformans protein extract and neither C. gattii isolate. Six proteins, including enolase, actin and thioredoxin reductase, were only found in the VGI extract. Thioredoxin peroxidase, an HSP70 family member, a 14-3-3 protein and pyruvate kinase were among the 12 proteins identied only in the VGII extract. The pathogen proteins identied by patient sera in early and advanced disease are not only potentially important in pathogenesis, but may also provide candidates for therapeutic intervention or prognostic markers. The identication of specic proteins by individuals infected with either VGI or VGII molecular types may be indicative of diversity in virulence mechanisms and provide a basis for tailor-made treatment regimens. Some of the more signicant proteins identied are discussed in greater detail below. Components of the thioredoxin antioxidant system, thioredoxin (Trx) and thioredoxin reductase (Trr), were specically identied using this model. In C. neoformans, Trx is important for intracellular survival, virulence in a mouse inhalation model and resistance to nitrosative stress.37 Trr is induced during oxidative and nitrosative stress, expressed during phagocytosis by macrophages, and is essential for the viability of C. neoformans.38 Trx and Trr were among eight proteins which reacted only with serum from the VGII-infected koala (C2). These ndings suggest that the thioredoxin antioxidant system is important for pathogenesis in VGII infection, and may even explain the reported increased virulence of this molecular type. Further investigation should focus on the specic role of this pathway in naturally occurring and laboratory models of cryptococcosis. Plants, fungi and bacteria possess a low molecular weight isoform of Trr, distinct from higher eukaryotes in both sequence and catalytic mechanisms,39 and therefore a potential therapeutic target. Pleurotin, an inhibitor of lowmolecular-weight-isoform Trr, has been used to effectively inhibit growth of the dermatophyte Trichophyton mentagrophytes in vivo and ex vivo, but had no effect on Candida albicans.40 The efcacy of pleurotin on systemic mycoses has not been investigated; however, the current results highlight its potential in cryptococcosis. Tsap1 and tsap3, both peroxiredoxins, were reactive with immune sera from clinical koalas only. Peroxiredoxins are important in defense against oxidative damage.41 Tsap1 is necessary for virulence in a mouse inhalation model and important for growth at 25 and 38.5 C and in the presence of oxidative and nitrosative stress.41 Both tsap1 and tsap3 are upregulated at 37 C and when exposed to hydrogen peroxide, but tsap3 is not essential for virulence.41 In Candida albicans, tsap1 is necessary for the induction of Trx and Trr expression in response to hydrogen peroxide.42 With established (clinical) disease and the development of a host immune response including oxidative and nitrosative attack by phagocytes, the up-regulation of pathways involved in surviving these host defenses could become more important for Cryptococcus to continue to cause disease and might explain the involvement of tsaps in late pathogenesis. Tsap1 may provide a novel target for therapeutic intervention in cryptococcosis. However, thiol-

Figure 5. 2-DE proles of (A) C. neoformans, (B) C. gattii (VGI), and (C) C. gattii (VGII). Numbered spots indicate proteins that were reactive with koala sera, the details of which can be found in Table 2. Supporting Information for each protein sequence identied can be found in Tables S2, S3 and S4 for C. neoformans, C. gattii (VGI), and C. gattii (VGII), respectively.

kinase, pyruvate dehydrogenase, elongation factor 1-beta and mannose-1-phosphate guanyltransferase) were also reactive with control koala sera (although reactivity was markedly lower in these individuals) and were excluded from further analyses. The immunodominant proteins identied (Table 2) included proteins involved in cellular respiration, cell cycle regulation, protein and amino acid metabolism, cell division and cell wall biosynthesis as well as several previously identied cryptococcal virulence factors and targets of the immune response in human and murine cryptococcosis. Thiol-specic antioxidant protein 1 (tsap1), tsap3 and a putative transferase were identied by both clinically affected animals only. Six proteins, including several heat shock proteins and enolase, were reactive with serum from the VGI-infected clinical koala only and 8 proteins, including thioredoxin, thioredoxin reductase and pyruvate kinase, reacted with the VGII-infected clinical koala only. In the subclinical group, only the 14-3-3 protein was identied by both koalas. Alpha-tubulin, ketol-acid reductoisomerase and ATP synthase, were recognized by sera from all affected koalas and a further 7 proteins were identied by at least one member
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specic antioxidant homologues also exist in mammals, so they may not provide a target that is suitably fungus-specic. Several HSPs were identied in our analyses, and a member of the HSP60 family was the only protein identied by patient sera in all three isolates. A specic role for HSPs in cryptococcal pathogenesis is undened, but HSP60 and 70 were upregulated in a rabbit model of cryptococcal meningitis44 and Ssa1, an HSP70 member, is required for C. neoformans virulence in a mouse model.45 Cryptococcal HSP70 members are well established immunogens30,46,47 and our ndings support this. Protective antibodies against microbial HSPs have been identied for systemic candidiasis48 and Mycograb, a recombinant antibody directed against an epitope of C. albicans HSP 90, improves or resolves the condition in a mouse model when used in conjunction with Amphotericin B (Amp B).49 Mycograb also acts in synergy with Amp B to reduce minimum inhibitory concentrations (MICs) against C. neoformans.50 If a protective role for anticryptococcal-HSP-antibodies can be elucidated, HSPs could provide targets for vaccine development. In patients with systemic candidiasis, a sustained antibody response to a 47 kDa fragment of Candida albicans HSP90 is correlated with a good prognosis.51 Host anticryptococcal-HSP antibodies may, therefore, have potential as prognostic indicators of a patients response to treatment. A number of proteins potentially important in tissue invasion52 were identied using this method (enolase, pyruvate kinase, ATP synthase, ketol-acid reductoisomerase and HSP60 and 70). In Candida albicans, enolase binds to human plasminogen and plasmin, inducing brinolysis and increasing its ability to cross a blood-brain barrier model.53 C. neoformans also binds plasminogen and converts it to plasmin in a cell wall-associated manner, increasing penetration in an extracellular matrix model.52 Pyruvate kinase, ATP synthase, ketol-acid reductoisomerase and HSP60 and 70 have been identied as plasminogen-binding proteins in C. neoformans.52 While enolase has not been conrmed as a plasminogen-binding protein in C. neoformans, it does possess a C-terminal lysine and is abundant in the cell wall.52 Sera from all affected koalas reacted with ketol-acid reductoisomerase and ATP synthase (subunit alpha), and enolase was among six proteins reactive with sera from the VGI-infected clinical koala only. Enolase may be a contributor to tissue invasion in C. gattii VGI infection, particularly in late pathogenesis. The identication of these proteins, as well as enolase, suggests that plasminogen-binding proteins may be important in early and late pathogenesis of cryptococcosis. Plasminogen-binding surface receptors may provide an alternate pathway for the development of novel diagnostic strategies in cryptococcosis. As in C. neoformans,30 enolase is immunodominant in Candida albicans,54 and this characteristic has enabled the development and successful use of an ELISA containing recombinant Candida enolase to diagnose invasive candidiasis.55 Similarly, an ELISA assay for enolase to diagnose progression from subclinical to clinical disease is likely to be a signicant part of future investigations. 14-3-3 proteins are evolutionarily and functionally conserved, participating in many cellular processes by binding to more than 200 different proteins.56 In this study, a cryptococcal 14-3-3 protein was immunodominant in both subclinical koalas but not clinical koalas. In other fungi, 14-3-3 proteins are essential for normal growth and virulence-associated morphogenesis.57,58 In cryptococcosis, 14-3-3 proteins are immunodominant in a mouse model of pulmonary disease30 and upregulated in a rabbit model
43 44

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of meningitis, and are also components of cryptococcal virulence bags.59 The reactivity of 14-3-3 proteins with sera from subclinically affected koalas suggests that they might be important in the early pathogenesis of cryptococcosis. Surprisingly, only one protein (a member of the HSP60 family) was detected by patient sera in all three cryptococcal strains. UDP-glucose 6-dehydrogenase and ATP synthase (subunit beta) were also identied in all three proteomes, but by control sera, indicating they likely recognized shared epitopes which are common to multiple organisms. Most of the remaining proteins were found in C. gattii only, with six and twelve of these identied only in molecular types VGI or VGII, respectively. The differences in identied proteins may reect variation in virulence pathways between the molecular types. This is supported by the fact that enolase, actin and elongation factors were only recognized by a VGI-infected koala in the VGI extract, and dihydrolipoyllysine-residue acetyltransferase, pyruvate kinase and ubiquinol-cytochrome C reductase complex core protein 2 were recognized only by the VGII-infected koala in the VGII protein extract.

Conclusion
There is an identied need to develop novel antifungal therapies for the treatment of mycoses, with a relatively small arsenal available to clinicians. Triazoles (e.g., uconazole, itraconazole) and the polyene Amphotericin B form the mainstay for treating cryptococcosis at present. Both classes of antimicrobials target the fungal membrane specic ergosterol synthesis pathway but have limitations of resistance and toxicity.60 Our results have highlighted several additional pathways, including the thioredoxin system and plasminogenmediated tissue invasion, which could be considered in the development of novel therapeutics. The fact that C. gattii possesses a thioredoxin reductase which is structurally distinct from the higher eukaryote form, might be of great value in targeting this virulence pathway. The candidates identied in this work should be considered further to determine their potential as targets for therapy. Several studies have examined the efcacy of anticryptococcal vaccines61,62 and therapeutic anticryptococcal antibodies,63,64 but the similarities between mammalian and fungal genomes, both being eukaryotes, make the elucidation of fungal-specic targets challenging. Our ndings have highlighted several cryptococcal proteins which are immunogenic in the koala model of natural disease, some of which were also immunodominant in a mouse model of experimental pulmonary cryptococcosis.30 Further work characterizing these proteins as vaccine targets could be valuable. While it is entirely possible that the proteins identied here may not produce a protective antibody response or induce a T cell immune response at all, dening these responses further could be valuable in assessing their suitability as vaccine targets. The ndings presented provide a good foundation for further investigation into the pathogenesis of cryptococcosis, using a natural model of disease. Extension of this work to conrm candidates as prognostic indicators would be valuable. Longitudinal studies of sera from koalas throughout the course of disease could be used to prole changes in antigen production by Cryptococcus, which might reect shifts in virulence pathways as more extensive tissue invasion occurs. Differences in antigen recognition associated with molecular type variation may explain the apparent variation in virulence between VGI and VGII molecular types. Investigation of changes to the host
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proteome in its response to cryptococcosis is a natural extension of this work to examine the host-pathogen interaction. Thirty-one cryptococcal proteins are identied in early and/ or late pathogenesis of cryptococcosis, using a novel protein extraction protocol and the koala immunome as a model for detecting disease-associated antigens. Several identied plasminogen-binding proteins may provide the mechanism by which Cryptococcus exerts its extensive tissue invasion and neurotropism. The thioredoxin system, by which Cryptococcus counteracts oxidative damage inicted by the host immune response, is clearly a component of cryptococcal pathogenesis in this model and may be a target for therapeutic intervention. A number of proteins uniquely identied by koalas infected with C. gattii VGI or VGII are worthy of investigation in relation to the different epidemiologies observed in these molecular types. Furthermore, the different proteins which were identied in the three strains might explain the observed differences in pathogenesis between these members of the Cryptococcus species complex. These ndings represent the rst examination of the full proteome of C. gattii and it is anticipated that these results will contribute to the knowledge of cryptococcal pathogenesis and enable the development of better treatment and prevention strategies for humans and animals.

Jobbins et al.
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Acknowledgment. The work was supported by an ARC Linkage grant (LP0560572) (with partners Australian Koala Foundation, The Koala Preservation Society of New South Wales, Symbion Vetnostics, Pzer Australia and WIRES), a Hermon Slade Foundation grant, and the methods development funded by an NHMRC grant (512399). We acknowledge Dr. Nathan Saul for molecular typing the clinical isolates. Paul Metcalfe, David Ward and Blackbutt Reserve, Mark Stone, Christie Brown and Billabong Wildlife Park, Leisa Denaro and Lamington Terrace Veterinary Surgery, as well as Helen McCutcheon and Melbourne Zoo provided access to clinical samples. For their intellectual support, we thank Associate Professor Dee Carter, Dr. Richard Malik, Professor Paul Caneld, Dr. Damien Higgins and the Koala Infectious Disease Research Group at The University of Sydney. Supporting Information Available: Table S1: Fungal isolates used in the study. Table S2: Protein sequence identications (C. neoformans). Table S3: Protein sequence identications (C. gattii, molecular type VGI). Table S4: Protein sequence identications (C. gattii, molecular type VGII). SI1: Analysis of peptides. SI2: Single peptide-based identications. This material is available free of charge via the Internet at http://pubs.acs.org. References
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