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Infection related stress adaptations in the secretome and wall proteome ofCandida albicans

Sorgo, A.G.

Publication date2013

Link to publication

Citation for published version (APA):Sorgo, A. G. (2013). Infection related stress adaptations in the secretome and wall proteomeof Candida albicans.

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Chapter 2

Mass spectrometric analysis of the secretome ofCandida albicans

Yeast (Chichester, England)

Sorgo, Alice_PROEF (all).ps Front - 18 T1 - Black CyanMagentaYellow

36

The pathogenic fungus Candida albicans secretes a considerable number of

hydrolases and other proteins. In-depth studies of the C. albicans secretome

could thus provide new candidates for diagnostic markers and vaccine

development. We compared various growth conditions differing in pH,

temperature, and the presence of the hyphal inducer N-acetylglucosamine. The

polypeptide content of the growth media was ca. 0.1 - 0.2% of the total biomass.

Using LC-tandem mass spectrometry, we identified 44 secretory proteins, the

transmembrane protein Msb2, 6 secretory pathway-associated proteins, and 28

predicted cytosolic proteins. Many secretory proteins are wall-related,

suggesting that their presence in the growth medium is at least partially due to

accidental release from the walls. Als3, Csa2, Rbt4, Sap4, and Sap6 were

enriched in the medium of hyphal cultures; Bgl2, Cht3, Dag7, Eng1, Pir1, Rbe1,

Scw11, Sim1/Sun42, Xog1, and Ywp1 in the medium of yeast cells; and Plb4.5

in pH 4-medium. Seven proteins (Cht3, Mp65, Orf19.5063/Coi1, Scw11, Sim1,

Sun41, and Tos1) were consistently present under all conditions tested. These

observations indicate that C. albicans tightly regulates its secretome. Mp65,

Sun41, and Tos1 were each predicted to contain at least one highly

immunogenic peptide. In total, we identified 29 highly immunogenic peptides

originating from 18 proteins, including two members of the family of secreted

aspartyl proteases. Fifty-six peptides were identified as proteotypic and will be

useful for quantification purposes. In summary, the number of identified

secretory proteins in the growth medium has been substantially extended, and

growth conditions strongly affect the composition of the secretome.

Sorgo, Alice_PROEF (all).ps Back - 18 T1 - Black CyanMagentaYellow

Candida albicans

37

Introduction

Candida albicans is commonly found as a benign commensal in many warm-

blooded animals. In humans it mainly resides on the skin and on mucosal

surfaces without causing significant harm to the host. But under certain

circumstances, such as a weakened host immune system, epithelial damage, or

disturbance of the microbial gut flora, C. albicans can cause superficial to deep-

seated mucosal infections. When the immune system of the host is severely

compromised, for example, during HIV infection, by chemotherapy or after

organ transplantation, the fungus can penetrate the tissue even further and gain

access to the bloodstream, which causes life-threatening systemic infections.

Several factors contribute to the pathogenic nature of Candida albicans (6). A

major virulence factor is the ability of C. albicans to switch from yeast to

hyphal growth. Loss of this ability leads to complete avirulence in a mouse

model of systemic infection (24). The wall and especially the covalently

anchored wall proteins are important contributors to pathogenicity as well. They

mediate adherence and invasion of host cells, promote biofilm formation, and

protect against the immune system (19). Secreted hydrolytic enzymes, like

lipases and proteases, play a role in tissue degradation. In addition, they

facilitate nutrient acquisition and invasion (37). Identification of other secreted

proteins could determine novel virulence factors. The identification of secreted

proteins could furthermore be of importance with regard to the development of

clinical markers or vaccine candidates.

Proteins in the growth medium that follow the classical secretory pathway are

marked for secretion by an N-terminal signal sequence. However, some proteins

have been identified in the medium of C. albicans that do not possess this

typical signal for secretion, raising the question if they reach the extracellular

space via an alternative route (29). A genome-wide computational study, based

on predicting the presence of a signal peptide, has identified 283 potentially

secreted proteins. Using mass spectrometric analysis of the secretome of C.


38

albicans grown under four different growth conditions, we identified 44

secretory proteins, a soluble form of the transmembrane protein Msb2, six

proteins predicted to be associated with compartments of the secretory pathway,

and 28 cytosolic proteins in the growth medium of Candida albicans, thereby

confirming and considerably extending earlier studies (15, 25, 47).

Additionally, we present evidence that many covalently anchored wall proteins

are partially released into the growth medium. We show further that the protein

composition of the secretome changes considerably in response to

environmental conditions. Interestingly, we identified a core set of seven

proteins that are found under all conditions tested. Most of the genes belonging

to this core set are highly conserved among other fungi, underlining their

functional importance. Finally, using the immunoinformatics algorithm POPI,

we identified a number of peptides that are predicted to elicit a strong response

in one or both arms of the immune response and thus might be valuable targets

for vaccine development.

Materials and methods Strains and growth conditions

Chemicals were obtained from Sigma-Aldrich unless otherwise stated. C. albicans

SC5314 (13) was used throughout this study. C. albicans was pre-cultured in liquid

YPD medium (10 g/l yeast extract, 20 g/l peptone and 20 g/l glucose) in a rotary shaker

at 200 rpm and 30°C overnight. The overnight culture was used to inoculate flasks

containing 50 ml YNBS (6.7 g/l YNB, 20 g/l sucrose, and 75 mM MOPSO [3-(N-

morpholino)-2-hydroxypropanesulfonic acid] set at pH 7.4 or 75 mM tartaric acid set at

pH 4 at an initial OD600= 0.05. These cultures were incubated for 18 h at 30 or 37°C and

shaken at 200 rpm. For hyphal induction, the culture medium was supplemented with 5

mM N-acetylglucosamine (GlcNAc).

Analysis of overnight cultures

For morphological analysis, the cells were visualized by light microscopy and

photographed. For determination of the biomass, the cultures were spun down after 18 h


Candida albicans

39

of growth and the pellet was dried at 60°C and weighed. To isolate the proteins from the

growth solution, the cultures were spun down and the supernatant was centrifuged again

to remove remaining cells. The medium was concentrated using 10-kDa cutoff filters

(Amicon Ultra-15 Centrifugal filter units, Millipore). The whole procedure was

performed at 4°C. After concentration, the medium proteins were quantified using the

BCA assay with bovine serum albumen as a standard (41). The amount of protein was

normalized to biomass dry weight.

Mass spectrometry of growth solution proteins

The concentrated proteins from the growth medium were reduced with 10 mM

dithiothreitol in 100 mM NH4HCO3 (1 h at 55 C). After cooling to room temperature,

the protein solutions were transferred to 10-kDa cut-off spin filter (Millipore) tubes and

centrifuged. The reduced proteins were alkylated with 65 mM iodoacetamide in 100

mM NH4HCO3 for 45 min at room temperature in the dark. The samples were quenched

were washed six times with 50 mM NH4HCO3 and either frozen in liquid nitrogen and

stored at -80 C or directly digested using 2 μg Trypsin Gold (Promega, Madison,WI)

from a 1 μg/μl stock solution for 18 h at 37°C. The tryptic digests were desalted using a

C18 tip column (Varian, Palo Alto, CA) according to the manufacturer’s instructions

and the peptide concentration was determined at 205 nm using a NanoDrop ND-1000

(Isogen Life Science, IJsselstein, The Netherlands) (9). Each sample was diluted with

0.1% trifluoroacetic acid to a final concentration of 75 ng/μl, and 10 μl per run were

injected onto an Ultimate 2000 nano-HPLC system (LC Packings, Amsterdam, The

Netherlands) equipped with a PepMap100 C18 reversed phase column (75 μm inner

diameter, 25 cm length; Dionex, Sunnyvale, CA). We used an elution flow rate of 0.3

μl/min along a linear gradient with increasing acetonitrile concentration over 45 min.

The eluting peptides were directly ionized by electrospray in a Q-TOF (Micromass,

Whyttenshawe, United Kingdom). Survey scans were acquired from m/z 350-1200. For

low energy collision-induced dissociation (MS/MS), the most intense ions were selected

in a data-dependent mode.

Analysis of mass spectrometric data

The generated spectra were processed using the MaxEnt3 algorithm included in the


40

Masslynx Proteinlynx software. The resulting pkl (peak list) files were submitted to an

internally licensed version of MASCOT (Matrix Science, Great Britain) using both a

complete C. albicans proteome database derived from the complete ORF translation

from the Candida Genome Database (2) and a dedicated database of GPI anchored and

signal peptide-containing proteins with 143 entries. To generate this database, a

preselected list of potential proteins was subjected to signal peptide prediction using

SignalP3.0 (3). In addition, all potential proteins were analyzed for the presence of a

GPI anchor sequence using the BIG-PI fungal predictor (10). Subsequently, predicted

N-terminal signal peptides and amino acids C-terminal of the predicted -amino acid

involved in GPI attachment were removed from the database. In MASCOT two

miscleavages and a tolerance of 0.6 Da for peptides and MS/MS were allowed.

Probabilistic MASCOT scoring was used to evaluate the identified peptides and

proteins. A P value of <0.05 was considered significant for peptide identification. Four

to six independently obtained biological samples were analyzed for each condition

(biological replicates). Each biological sample was subjected to three runs with MS/MS

selection switching times of 2 s, 1.5 s, and 1.25 s, respectively. For a semi-quantitative

analysis of our data, we calculated for each growth condition the mean of the total

number of peptide identifications per biological replicate.

Peptides identified in at least 50% of all runs in a single condition were considered to be

proteotypic (27) (see also Supplemental Table S5 and S6 for details). The

immunogenicity of peptides was predicted using the POPI 2.0 web server (50).

All Supplemental materials can be found online under the following link:

http://onlinelibrary.wiley.com/doi/10.1002/yea.1775/suppinfo

Results and discussion

Selection of growth conditions for secretome analysis

As C. albicans is known to acidify its environment (49), a stable pH requires

buffering of the culture medium. For cultures grown at pH 4, we selected

tartaric acid as a buffer. It is not metabolized by C. albicans (data not shown)

and has two pKa values, one at 3.01 and the other at 4.37, which makes it

suitable for buffering at pH 4. To buffer cultures at pH 7.4, we used MOPSO,

which has a pKa of 6.90 at 37ºC. It is a hydrophilic molecule, which minimizes


Candida albicans

41

its uptake by the cell in its uncharged form. We used buffer concentrations of 75

mM, which kept the pH stable throughout the overnight culturing period used

(data not shown). Elevated glucose concentrations inhibit hyphal growth and

activate the glucose repression pathway leading to the repression of many

genes, including several genes involved in the utilization of alternative carbon

sources (26, 36). To avoid glucose repression, we used sucrose as a carbon

source. Sucrose does not affect hyphal growth and allows the expression of

glucose-repressed genes such as HEX1, which encodes a secretory N-

acetylglucosaminidase (33), and MAL2, which encodes an -glucosidase

(maltase) (12). Importantly, in contrast to S. cerevisiae, C. albicans does not

secrete an invertase to hydrolyze sucrose extracellularly but uses a H+/sucrose

symporter to transfer it over the plasma membrane, and hydrolyzes it

intracellularly using Mal2 (53).

Table1. Morphology, biomass production, and medium protein concentration of overnight cultures grown under various conditions. Averages of at least three cultures are shown (Coefficient of Variation 25%). Growth conditiona

Morphology of overnight cultures

Biomass mg/ml

Medium protein g/mg biomass

pH 7.4, 37ºC Yeast and hyphae 2.2 0.83 pH 7.4, 37ºC + 5 mM GlcNAc

Mainly hyphae 2.0 2.32

pH 7.4, 30ºC Exclusively yeast cells 2.8 0.98 pH 4.0, 37ºC Exclusively yeast cells 3.3 0.87 aYNB containing 2% sucrose as carbon source.

To establish if the composition of the C. albicans secretome depends on growth

conditions, we selected conditions that were expected to result in a distinct

protein expression and morphology (Table 1) (4, 14, 39, 45, 52). When grown

overnight at pH 7.4 and at 37°C the culture consisted of a mixture of yeast and

hyphal cells. In the presence of GlcNAc, hyphal growth predominated (data not

shown). When the temperature was decreased to 30°C or the medium pH

lowered to 4, the cultures consisted exclusively of yeast cells.


42

After concentration of the C. albicans culture supernatants, their protein

contents were determined and normalized to the biomass of the cultures. Table

1 shows that growth was optimal at pH 4 and 37ºC and that hyphal growth

resulted in diminished biomass formation. Except for the GlcNAc-

supplemented culture, the protein content of the culture supernatants was

similar (0.8 – 1.0 g protein/mg biomass (dry weight) corresponding to about

0.1% of the total biomass). The growth medium of GlcNAc-induced cultures

contained about 2.3 g protein/mg biomass, suggesting that hyphae either

secrete more proteins or are more sensitive to shearing forces occurring in

shaken cultures.

Composition of the Candida albicans secretome

The number of predicted secretory proteins (proteins with an N-terminal

secretory signal sequence but without an internal transmembrane sequence and

mitochondrial signal sequence) for C. albicans is 333 (21), including 50

predicted GPI proteins, which are believed to become tail-anchored to the

plasma membrane or the cell wall (8). Under our growth conditions, we

identified a total of 44 secretory medium proteins. Interestingly, twenty-nine of

them have a cell wall-related function or location, including eighteen GPI-

proteins that have been described as being covalently anchored to the cell wall

(19). This is consistent with earlier studies of C. albicans and Candida glabrata

in which also known GPI wall proteins were found in the culture solution (44).

As our cultures were shaken at 200 rpm, it seems possible that non-covalently

bound wall-associated proteins such as Bgl2, Cht1, Cht3, Eng1, and Xog1 are

partially washed out of the walls. Wall proteins known to be covalently bound

to the wall polysaccharides, such as GPI proteins, Pir1, Scw11, Sim1/Sun42,

and Tos1, might also be partially washed out, namely, after their release from

the plasma membrane but before their covalent attachment to skeletal

polysaccharides. Release might also take place during cell wall remodeling, for


Candida albicans

43

example, during isotropic growth. This hypothesis is supported by the

identification of -1,3-glucan-associated forms of the wall proteins Als3 and

Hyr1 in the supernatant of C. albicans cultures (48). In addition to the wall-

related proteins, fifteen secretory proteins were identified without a known

wall-related function or location, including five members of the Sap (secreted

aspartyl protease) family, a glucoamylase, a hexosaminidase, and a

phospholipase. A fragment of Msb2, a transmembrane protein located in the

plasma membrane (for further discussion see below), and six proteins predicted

to be associated with intracellular compartments of the secretory pathway were

also found. Finally, 28 predicted cytosolic proteins were identified; many of

which are known to be highly abundant intracellularly (Supplementary Tables

S1-4) (20).

Proteotypic peptides are defined as peptides that are observed in at least 50% of

all corresponding protein identifications in mass spectrometry-based

proteomics; they are responsible for the majority of protein identifications (27).

Since we performed 12-18 LC-MS/MS runs per condition, we have a sound

basis to recognize proteotypic peptides. We identified 56 proteotypic peptides in

23 proteins with up to 6 proteotypic peptides per protein (Supplemental Tables

S5 and S6). An important use of proteotypic peptides is absolute quantification

of proteins (27).

Dynamics of the Candida albicans secretome

Because the total number of peptide identifications of a particular protein is

correlated with the concentration of that protein (22), we calculated for each

protein the average number of peptide identifications/biological replicate under

the four growth conditions studied (Table 2). This semi-quantitative approach

shows that different modes of growth such as yeast or hyphal growth or certain

conditions such as low pH lead to clear differences in the protein composition

of the growth solution (Tables 2 and 3). Seven proteins (Cht3, Mp65,


44

Orf19.5063/Coi1, Scw11, Sim1/Sun42, Sun41, and Tos1) were found under all

four growth conditions tested when “the mean 1 in all four conditions” was

used as the threshold value. Notice, however, that the concentrations of Cht3,

Scw11, and Sim1/Sun42 were considerably decreased in hyphal cultures.

Importantly, we identified almost all predicted tryptic peptides of Mp65,

Sim1/Sun42, and Sun41 that were in the detection range of our instrument,

suggesting that they are abundant medium proteins. This is also consistent with

their high Codon Adaptation Index values of 0.65, 0.70, and 0.61, respectively

(Candida Genome Database, http://www.candidagenome.org).

Table 2. Mass spectrometric analysis of the proteins released into the growth medium under various environmental conditions. Y, exclusively yeast cells; H, predominantly hyphal growth; Y + H, both growth forms present in considerable amounts. Protein Properties and functiona No. of

PPb

Mean of peptide identifications in the biological replicatesc

pH 7.4 37°C Y+H

pH 7.4 37°C +GN

H

pH 7.4 30°C

Y

pH 4 37°C

Y Wall-related proteins Als3 GPI-WP, adhesin 3 1.0 6.5 0 0 Als4 GPI-WP, adhesin 0 0 0 0.2 0 Bgl2 Transglucosylase 2 3.0 0.3 4.2 7.4 Cht1 Chitinase 1 0 0 2.8 3.0 Cht2 GPI-WP, chitinase 1 0.8 3.7 2.8 1.6 Cht3 Chitinase 3 8.2 1.3 10.2 8.4 Crh11 GPI-WP, transglycosylase 0 0.4 0 0 0 Ecm33 GPI-WP, wall integrity 0 2.0 0 0.8 1.6 Eng1 Endo-1,3- -glucanase 3 3.8 0 8.0 5.6 Fgr41 Pga35, GPI protein 0 0.2 0 0.2 1.4 Mp65 Transglycosylase 6 13.2 10 14.5 15.4 Pga4 GPI-WP, transglucosidase 1 0.8 0 1.0 2.0 Pga45 GPI-WP, unknown 0 0.4 0.3 0.2 0.4 Phr1 GPI-WP, transglucosidase 0 0.2 0 0 0 Pir1 Wall cross-linking protein 1 1.2 0 1.5 3.6 Rbt1 GPI-protein, Flo11

domain 0 0.2 0 0 0

Rbt5 GPI-WP, acquisition of hemoglobin

1 0.6 0.3 2.5 1.8

Rhd3 Pga29, GPI-WP 0 0 0 0 0.4 Sap10 GPI-WP, protease 0 0 0 0 0.8 Scw11 1,3- -Glucanase 5 10.6 1.3 10.2 8.4


Candida albicans

45

Sim1 Sun42, wall maintenance 3 7.0 1.8 9.2 11.6 Sod4 GPI-WP, superoxide

dismutase 0 0.2 0 0 0

Sod5 GPI-WP, superoxide dismutase

0 0 0.7 0 0

Ssr1 GPI-WP, wall structure 1 1.2 0 0 2.8 Sun41 Wall maintenance 4 9.2 6.0 10 12.2 Tos1 Unknown 6 10.4 8.7 11.8 13.6 Utr2 GPI-WP, glycosidase 3 3.4 0.5 0 8.0 Xog1 Exo-1,3- -glucanase 2 10 0.8 10 7.6 Ywp1 GPI-WP 0 3.6 0 2.2 2.6 Other proteins Ape2d Metallo-aminopeptidase 0 0 0.2 0 0 Csa2 CFEM domain 0 0.6 1.7 0 0 Cyp5d Peptidyl-prolyl cis-trans

isomerase 0 0.2 0.7 0.2 0

Dag7 Unknown 1 1.8 0 2.8 3.8 Gca1 Glucosyl hydrolases 0 0.4 0 0.2 1.2 Hex1 Hexosaminidase 0 0.2 0 0 0 Kar2 d Chaperone-like protein 0 0 0.7 0 0 Mnt1d 1,2-mannosyl transferase 0 0.4 0 0 0 Msb2 Putative sensor protein 0 2.0 0.7 1.2 0.2 Op4 Opaque-specific 0 0 0 0 0.8 Orf19. 5063e

Ciclopirox Olamine-Induced

3 7.0 3.3 7.2 1.6

Pdi1d Protein disulfide-isomerase

0 1.0 0 0 0

Plb4.5 GPI-protein, phospholipase

1 0 0 0 6.0

Pra1 Immune evasion protein 0 0.2 0 0 0 Pry1 Opaque-specific,

SCP_PRY_like domain 0 0 0 0 0.4

Rbe1 SCP_PRY_like domain 0 1.6 0 3.5 1.2 Rbt4 SCP_PRY_like domain 3 0.8 6.8 2.0 0.2 Sap4 Secreted aspartyl protease 1 0 6.2 0 0 Sap5 Secreted aspartyl protease 0 0.2 0.3 0 0 Sap6 Secreted aspartyl protease 1 0 9.2 0 0 Sap8 Secreted aspartyl protease 0 0 0 0 0.6 Ubi3 0 0.6 1.7 0 0 a CGD, Candida Genome Database; GPI-WP, GPI-anchored wall protein. b PP = Proteotypic peptide; For sequences see Supplemental Table S6. c Mean of the total number of peptide identifications in the biological replicates (n = 4-6) see also Supplementary Tables S1-S4. d Predicted to be ER- and Golgi-associated proteins. e Proposed name: Coi1. GN= GlcNAc; Y=Yeast morphology; H=Hyphal morphology.


46

Table 3 further shows that 4-5 proteins are enriched in the growth medium of a hyphal culture (Als3, Rbt4, Sap4, Sap6, and presumably also Csa2). The transcript levels of PIR1, RBE1, and YWP1 rapidly and strongly decline and remain low when YPD-grown yeast cells are transferred to a hyphal induction medium (42). Consistent with this, peptides of Pir1, Rbe1, and Ywp1 are absent in the growth medium of hyphal cultures (GlcNAc-induced hyphal growth) but present in the media of yeast(-containing) cultures. In total, ten proteins are enriched in the media of yeast-containing cultures (Bgl2, Cht3, Dag7, Eng1, Pir1, Rbe1, Scw11, Sim1/Sun42, Xog1,Ywp1). The medium of pH 4-grown cells is clearly enriched in Plb4.5 and probably also, but to a lesser extent, in Fgr41/Pga35, the opaque phase-specific proteins Op4 and Pry1, and the aspartyl proteases Sap8 and Sap10. These observations illustrate the dynamic nature of the secretome. Table 3. Relative enrichment of proteins in the growth solution under various environmental conditions. Conditionsa Characteristic features Secretory proteins All Mp65, Cht3, Orf19.5063/Coi1,

Scw11, Sun41, Tos1b 2 Predominantly hyphae Als3, Csa2, Rbt4, Sap4, Sap6 1+3+4 Mixed culture or

exclusively yeast cells Bgl2, Cht3, Dag7, Eng1, Pir1, Rbe1, Scw11, Sim1, Xog1, Ywp1

4 pH 4 Plb4.5 aGrowth conditions: 1: pH 7.4, 37°C; 2: pH 7.4, 37°C, +5 mM GlcNAc; 3: pH 7.4, 30°C; 4: pH 4, 37°C. bProteins with an average total peptide score in the biological replicates 1.

Discussion of individual secretory proteins in the growth solution

Csa2 is a small secretory protein without a GPI-anchor signal sequence. It has a

CFEM domain, but its function is unknown. Interestingly, it seems to be

enriched in hyphal cultures (Table 3). Fgr41 (Pga35) is a predicted GPI protein;

the encoding gene has been annotated as a possibly spurious ORF (CGD). We

have identified Fgr41/Pga35 in the growth medium in three of the four

conditions tested, showing that the corresponding gene is a genuine ORF.


Candida albicans

47

Gca1 is a predicted glucoamylase, which is consistent with the observation that

C. albicans can grow on soluble starch as a carbon source (31). Conceivably, it

might also allow C. albicans to use host glycogen as a carbon source.

Hex1 is an N-acetylglucosaminidase. When GlcNAc is used as a carbon source,

Hex1 activity is strongly induced (32). As Hex1 is only found in the growth

medium under a single growth condition (pH 7.4, 37°C) and not in GlcNAc-

supplemented medium, it seems likely that the presence of sucrose prevents the

induction of Hex1 and that GlcNAc primarily acts a hyphal-inducing compound

and does not need to be metabolized for this effect to occur.

Mp65 is found in all four growth media and its level does not seem to change

significantly. This is in agreement with earlier observations indicating that

MP65 is constitutively expressed during yeast and hyphal growth (42).

Msb2 possesses an N-terminal signal peptide. It is described as a sensor of cell

wall damage and is predicted to be a transmembrane protein associated with the

plasma membrane (35). We identified five different peptides that are unique for

Msb2 in tryptic digests of medium proteins, indicating that Msb2 or a processed

form of Msb2 is also present in the culture medium. This is consistent with the

observations by Vadaie and co-workers in S. cerevisiae who found that Msb2 is

processed into medium and cell-associated forms. As a result, a large fragment

of ScMsb2 ends up in the growth medium (51). Interestingly, the peptides

identified by us in CaMsb2 are located closely to each other (1154-1177, 1212-

1219, 1223-1237, 1238-1253, 1254-1290) in a region that is homologous to the

so-called cleavage domain of ScMsb2, consistent with the notion that CaMsb2

and ScMsb2 are functionally related (51).

Op4 and Pry1 are found only in the medium of pH 4-grown cells. Both have

been described as opaque cell-specific, but in view of our results it cannot be

excluded that they are also synthesized by white-phase cells at low pH.

Orf19.5063 is a predicted protein in Assemblies 19, 20, and 21 of the Candida

genome. We found it in the culture medium under all four growth conditions.


48

Interestingly, Orf19.5063 is upregulated in the presence of the antifungal

compound ciclopirox olamine, which causes a transcriptional profile resembling

that of iron restriction (38). Therefore we propose the name Coi1 (Ciclopirox

Olamine Induced 1) for Orf19.5063. Intriguingly, Coi1 is conserved only in C.

albicans, C. dubliniensis, C. tropicalis and Lodderomyces elongisporus, which

have all been implicated in causing bloodstream infections in humans.

Plb4.5 is a putative phospholipase B and is predicted to be GPI-anchored. It is

only found in the medium of pH 4-grown cells. Under these conditions nine

different peptides were identified suggesting that it might be an abundant

protein.

Pry1, Rbe1, and Rbt4 share a conserved domain (SCP_PRY1_like domain), but

the function of this domain is unknown. Rhd3 is a GPI wall protein that is also

known as Pga29. We detected it only in the medium of pH 4-grown cells. It

does not seem to be strictly correlated with yeast growth because it was absent

in a yeast culture growing at pH 7.4 and 30ºC (Table 2).

The Sap family, which has been extensively investigated, consists of ten

members, two of which are wall-bound GPI proteins (Sap9 and Sap10), whereas

the others are secreted into the growth medium (30). The subfamily SAP4 to

SAP6 is known to be coordinately expressed with hyphal growth and in

accordance with this we found Sap4 and Sap6 (and to a much lesser extent

Sap5) in the medium of hyphal cultures, but not in the media of yeast cultures

(Tables 2 and 3). Interestingly, Sap 8 and the GPI-modified Sap10 are only

found in pH 4-grown cell cultures. The Sap family thus represents a clear

example of the adaptability of the secretome.

Sim1 (Sun42) and Sun 41 both belong to the Sun family and share the so-called

SUN domain. The transcript levels of SIM1/SUN42 strongly decrease during

hyphal growth (11, 17), consistent with our observation that the level of Sim1 in

the medium of a predominantly hyphal culture is much lower than in

(predominantly) yeast cultures.


Candida albicans

49

Finally, Ape2, Cyp5, Kar2, Mnt1, and Pdi1 each possess a predicted N-terminal

signal peptide or a transmembrane domain close to the N-terminus and are

believed to be associated with the endoplasmic reticulum or the Golgi apparatus

(5, 40).Their presence in the medium is consistent with the observation that the

culture medium of C. albicans contains membrane vesicles (1). Table 2 shows

that they are mainly found in the medium of (partially) hyphal cultures, and are

almost completely absent in yeast cultures, suggesting that their presence in the

medium is due to hyphal breakage. Intriguingly, Ubi3 also possesses a predicted

N-terminal signal peptide or N-terminal transmembrane domain, raising the

question whether this predicted ribosomal protein might be associated with the

rough endoplasmic reticulum.

Identification of peptides with high immunogenic potential

All identified tryptic peptides were subjected to a prediction of their potential to

induce the proliferation of cytotoxic (CTL) and helper T-cell lymphocytes

(HTL) (Table 4) (50). In our complete set of 228 peptides, 29 (~13%) show a

high immunogenic potential, while 77 (~34%) show no immunogenic potential

at all whereas the remainder had an intermediate potential (Table 4,

Supplemental Table S6).

Mp65, which seems to be an abundant medium protein under all growth

conditions tested, also contains a predicted highly immunogenic peptide

(LYGVDCDQVSAVLK). This result was consistent with the predicted

antigenicity of this peptide by another algorithm

(http://bio.dfci.harvard.edu/Tools/antigenic.html). Based on mouse vaccination

studies, Mp65 seems indeed a promising vaccine candidate (34). Sim1/Sun42

and Sun 41 are functionally redundant and the double deletant is nonviable (11).

As the SUN domain of both proteins contains peptides with a high predicted

immunogenicity (Table 4), this domain also seems an attractive vaccine

candidate.


50

Table 4. Predicted immunogenicity of secretory protein peptides. Protein Sequence (position) Immunogenicitya

CTLb HTLc Als3/5 APFTLR (306-311) 3 0

Cht2 LSSAIEEIK (259-267) 3 1 Eng1 ELAANIAATVK (797-807)

DASNPSADDTYFPVSR (932-947) 2 3

3 1

Gca1 LNVHIEPTDLTDVFVLPEELVVKPK (108-132) GHSITGLGESIHGSLNEPGVVK (193-214) YFDNPVHPPFEVGYSGSDYPLGFDK (479-503)

3 0 3

0 3 0

Mp65 LYGVDCDQVSAVLK (161-174) 1 3 Msb2 SALNYPFVVENSISSAQIFQYLPR (1154-1177)

ALGSFITTPGSAIYR (1223-1237) 3 3

3 2

Pga4 AGIYVILDVNTPHSSITR (99-116) 3 1 Pga45 DANTEQTIEGILK (86-98) 0 3 Rbt4 LGCAYK (312-317)

SYMAENVLRPQ (348-358) 2 2

2 2

Sap4/6 TLSVGLR (269-275, 270-276) 0 3 Sap4 LSVIVDTGSSDLWVPDSNAVCIPK (102-125)

YADGSVAQGNLYQDTVGIGGVSVR (159-182) 0 0

3 3

Sap6 GNLYQDTVGIGGASVK (168-183) 0 3 Sim1 TDYPGSENMNIPTLLSAGGK (233-252)

TSTQYYVNNAGVSVEDGCIWGTEGSGVGNWAPVVLGSGTTGGK (271-313)

3 0

3 3

Sod5 HGNIMGESYK (118-127) TEYDDSYISLNEK (128-140)

2 2

2 3

Sun41 IVGESGSTVSGSCSYANGK (376-394) 0 3 Tos1 DSYYTPGSTDNCVFLNYHGGSGSGVWSAK (231-259) 0 3 Utr2 EIYATAYDIPNDVK (297-310) 2 2 Xog1 QISNLGLNFVR (120-130)

DSYNFQNGDNTQVTLNVLNTIFK (189-211) QFFLDGYNSLR (243-253)

3 2 3

2 2 1

Ywp1 VINVPAR (87-93) 3 0 a POPI algorithm (50); 3: PD50 < 1 nM; 2: PD50 = 100 nM - 1 nM; 1: PD50 = 10 mM - 100 nM; 0: PD50 >10 mM, in which PD50 = Protective Dose that protects 50% of the animals challenged b Cytotoxic T-lymphocytes, c Helper T-lymphocytes

Concluding remarks

We have analyzed the proteins of C. albicans that are released into the growth

medium under various environmental conditions. This allowed us to compare

yeast and hyphal cultures and the effect of pH. The polypeptide content of the


Candida albicans

51

growth medium was 0.1% of the total biomass for three of the four conditions

tested, but was >2-fold higher in the hyphal culture (Table 1). In total we

identified 44 secretory proteins, which is considerably more than in earlier

studies. Twenty-nine of them had a wall-related location or function, whereas

15 possessed a function unrelated to the cell wall. We also detected a soluble

form of the plasma membrane sensor protein Msb2 and six proteins predicted to

be associated with early compartments of the secretory pathway. Conceivably,

some of the wall-related proteins found in the culture solution could be involved

in biofilm formation and, in particular, in the formation of extracellular

polysaccharides (15). In total, we detected 28 predicted cytosolic proteins in the

various culture solutions (Supplementary Tables S1-4). Interestingly, in pH 4-

grown yeast cultures we did not identify any cytosolic protein whereas in the

hyphal culture we identified considerably more cytosolic proteins than in the

other cultures, suggesting that in comparison to yeast cells hyphae are more

liable to breakage due to the shearing forces inherent to shaking flask cultures.

We cannot exclude that medium proteins are degraded over time. However, we

found many more tryptic peptides than semitryptic peptides (peptides that match

trypsin specificity only at one terminus), suggesting that proteolytic degradation

was limited (data not shown). Albuquerque and co-workers have described

membrane-bound vesicles in the culture medium of Histoplasma capsulatum

and other ascomycetous fungi including C. albicans (1). Interestingly, when the

culture medium is filtered using a 200-nm filter, the number of cytosolic

proteins in the filtrate is negligible whereas the number of secretory proteins

does not seem to be affected (unpublished data). This suggests that the cytosolic

proteins found in the medium are contained in membranous vesicles (1).

Compared to the predicted secretomes (proteins with an N-terminal secretory

signal peptide, including GPI-proteins but excluding proteins with an internal

transmembrane sequence) of other yeasts such as Kluyveromyces lactis (178

proteins) (46), Saccharomyces cerevisiae (163 proteins), Pichia pastoris (88


52

proteins) (28), Schizosaccharomyces pombe (66 proteins) (23), C. albicans has

a relatively large predicted secretome. Possibly, the secretomes of non-

pathogenic yeasts tend to be smaller as has also been observed for plant

pathogenic mycelial fungi (7). Alternatively, the higher number of predicted

secretory proteins might be related to the pleomorphic nature of C. albicans.

Importantly, of the 44 secretory proteins we were able to identify in the growth

medium, only Mp65, Sun41, and Tos1 seemed to be consistently and

abundantly present under all four conditions. This observation is supported by

previous studies (15, 25). The corresponding genes are highly conserved in

many fungi, underlining their functional importance. These proteins could

therefore be useful for the development of clinical markers or as vaccine

candidates. Interestingly, a recently developed algorithm for the identification

of immunogenic peptides predicts that Mp65, Sun41, and Tos1 each possess a

highly immunogenic peptide (50).

The composition of the secretome differed considerably between pH 4- and pH

7.4-grown cells and between yeast and hyphal cultures (Tables 2 and 3). This

clearly demonstrates that the composition of the secretome can vary widely. If

our assumption is correct that some of the medium proteins are actually washed-

out wall proteins, the dynamic nature of the wall proteome of C. albicans (16,

43) could partially explain the observed differences. However, we also observed

relative enrichment of specific hydrolytic enzymes in the medium depending on

growth conditions and morphology (Table 3). This is illustrated by the increased

levels of Sap4 and Sap6 in hyphal culture medium and of Plb4.5 at pH 4 (to a

lesser extent the levels of Gca1 and Sap8 also seem to increase at pH 4; see

Table 2). The capability of adapting the composition of the secretome probably

helps C. albicans to better cope with the different growth conditions that it

encounters on the various sites of infection and during the subsequent infection

process. The proposed tight regulation of the composition of the secretome by

C. albicans probably also explains, at least to some extent, why only a limited


Candida albicans

53

number of the predicted secretory proteins have been identified so far. For

example, we did not identify any member of the extended family of secretory

lipases (18). The most likely explanation is that our growth conditions and in

particular the use of sucrose instead of lipids as a carbon source did not favor

the expression of the lipase genes (18).

Acknowledgements

We thank Dr. Leo de Koning for his stimulating support and advice. F.M.K.

acknowledges the financial support by the EU Program FP7-214004-2

FINSysB.

References 1. Albuquerque, P. C., E. S. Nakayasu, M. L. Rodrigues, S. Frases, A. Casadevall, R. M. Zancope-Oliveira, I. C. Almeida, and J. D. Nosanchuk. 2008. Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes. Cell Microbiol 10:1695-1710. 2. Arnaud, M. B., M. C. Costanzo, M. S. Skrzypek, P. Shah, G. Binkley, C. Lane, S. R. Miyasato, and G. Sherlock. 2007. Sequence resources at the Candida Genome Database. Nucleic Acids Res 35:D452-456. 3. Bendtsen, J. D., H. Nielsen, G. von Heijne, and S. Brunak. 2004. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783-795. 4. Bensen, E. S., S. J. Martin, M. Li, J. Berman, and D. A. Davis. 2004. Transcriptional profiling in Candida albicans reveals new adaptive responses to extracellular pH and functions for Rim101p. Mol Microbiol 54:1335-1351. 5. Buurman, E. T., C. Westwater, B. Hube, A. J. Brown, F. C. Odds, and N. A. Gow. 1998. Molecular analysis of CaMnt1p, a mannosyl transferase important for adhesion and virulence of Candida albicans. Proc Natl Acad Sci U S A 95:7670-7675. 6. Calderone, R. A., and W. A. Fonzi. 2001. Virulence factors of Candida albicans. Trends Microbiol 9:327-335. 7. Choi, J., J. Park, D. Kim, K. Jung, S. Kang, and Y. H. Lee. 2010. Fungal Secretome Database: Integrated platform for annotation of fungal secretomes. BMC Genomics 11:105. 8. De Groot, P. W., K. J. Hellingwerf, and F. M. Klis. 2003. Genome-wide identification of fungal GPI proteins. Yeast 20:781-796. 9. Desjardins, P., J. B. Hansen, and M. Allen. 2009. Microvolume spectrophotometric and fluorometric determination of protein concentration. Curr Protoc Protein Sci Chapter 3:Unit 3 10. 10. Eisenhaber, B., G. Schneider, M. Wildpaner, and F. Eisenhaber. 2004. A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans,


54

Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe. J Mol Biol 337:243-253. 11. Firon, A., S. Aubert, I. Iraqui, S. Guadagnini, S. Goyard, M. C. Prevost, G. Janbon, and C. d'Enfert. 2007. The SUN41 and SUN42 genes are essential for cell separation in Candida albicans. Mol Microbiol 66:1256-1275. 12. Geber, A., P. R. Williamson, J. H. Rex, E. C. Sweeney, and J. E. Bennett. 1992. Cloning and characterization of a Candida albicans maltase gene involved in sucrose utilization. J Bacteriol 174:6992-6996. 13. Gillum, A. M., E. Y. Tsay, and D. R. Kirsch. 1984. Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S.cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet 198:179-182. 14. Gow, N. A. 2009. Fungal morphogenesis: some like it hot. Curr Biol 19:R333-334. 15. Hiller, E., S. Heine, H. Brunner, and S. Rupp. 2007. Candida albicans Sun41p, a putative glycosidase, is involved in morphogenesis, cell wall biogenesis, and biofilm formation. Eukaryot Cell 6:2056-2065. 16. Hoyer, L. L., T. L. Payne, M. Bell, A. M. Myers, and S. Scherer. 1998. Candida albicans ALS3 and insights into the nature of the ALS gene family. Curr Genet 33:451-459. 17. Hube, B., M. Monod, D. A. Schofield, A. J. Brown, and N. A. Gow. 1994. Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Mol Microbiol 14:87-99. 18. Hube, B., F. Stehr, M. Bossenz, A. Mazur, M. Kretschmar, and W. Schafer. 2000. Secreted lipases of Candida albicans: cloning, characterisation and expression analysis of a new gene family with at least ten members. Arch Microbiol 174:362-374. 19. Klis, F. M., G. J. Sosinska, P. W. de Groot, and S. Brul. 2009. Covalently linked cell wall proteins of Candida albicans and their role in fitness and virulence. FEMS Yeast Res 9:1013-1028. 20. Kusch, H., S. Engelmann, R. Bode, D. Albrecht, J. Morschhauser, and M. Hecker. 2008. A proteomic view of Candida albicans yeast cell metabolism in exponential and stationary growth phases. Int J Med Microbiol 298:291-318. 21. Lee, S. A., S. Wormsley, S. Kamoun, A. F. Lee, K. Joiner, and B. Wong. 2003. An analysis of the Candida albicans genome database for soluble secreted proteins using computer-based prediction algorithms. Yeast 20:595-610. 22. Liu, H., R. G. Sadygov, and J. R. Yates, 3rd. 2004. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem 76:4193-4201. 23. Liu, Y. L., Y. F. Liu, and J. P. Xie. 2007. Characterization of Schizosaccharomyces pombe secreted proteins. Yi Chuan 29:250-256. 24. Lo, H. J., J. R. Kohler, B. DiDomenico, D. Loebenberg, A. Cacciapuoti, and G. R. Fink. 1997. Nonfilamentous C. albicans mutants are avirulent. Cell 90:939-949. 25. Maddi, A., S. M. Bowman, and S. J. Free. 2009. Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans. Fungal Genet Biol 46:768-781. 26. Maidan, M. M., J. M. Thevelein, and P. Van Dijck. 2005. Carbon source induced yeast-to-hypha transition in Candida albicans is dependent on the presence of


Candida albicans

55

amino acids and on the G-protein-coupled receptor Gpr1. Biochem Soc Trans 33:291-293. 27. Mallick, P., M. Schirle, S. S. Chen, M. R. Flory, H. Lee, D. Martin, J. Ranish, B. Raught, R. Schmitt, T. Werner, B. Kuster, and R. Aebersold. 2007. Computational prediction of proteotypic peptides for quantitative proteomics. Nat Biotechnol 25:125-131. 28. Mattanovich, D., A. Graf, J. Stadlmann, M. Dragosits, A. Redl, M. Maurer, M. Kleinheinz, M. Sauer, F. Altmann, and B. Gasser. 2009. Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris. Microb Cell Fact 8:29. 29. Monteoliva, L., M. L. Matas, C. Gil, C. Nombela, and J. Pla. 2002. Large-scale identification of putative exported proteins in Candida albicans by genetic selection. Eukaryot Cell 1:514-525. 30. Naglik, J. R., S. J. Challacombe, and B. Hube. 2003. Candida albicans secreted aspartyl proteinases in virulence and pathogenesis. Microbiol Mol Biol Rev 67:400-428, table of contents. 31. Nickerson, W. J., and Z. Mankowski. 1953. A polysaccharide medium of known composition favoring chlamydospore formation in Candida albicans. J Infect Dis 92:20-25. 32. Niimi, K., M. Niimi, M. G. Shepherd, and R. D. Cannon. 1997. Regulation of N-acetylglucosaminidase production in Candida albicans. Arch Microbiol 168:464-472. 33. Niimi, M., A. Kamiyama, M. Tokunaga, and H. Nakayama. 1987. Evidence for a glucose effect on N-acetylglucosamine catabolism in Candida albicans. Can J Microbiol 33:345-347. 34. Pietrella, D., P. Lupo, A. Rachini, S. Sandini, A. Ciervo, S. Perito, F. Bistoni, and A. Vecchiarelli. 2008. A Candida albicans mannoprotein deprived of its mannan moiety is efficiently taken up and processed by human dendritic cells and induces T-cell activation without stimulating proinflammatory cytokine production. Infect Immun 76:4359-4367. 35. Roman, E., F. Cottier, J. F. Ernst, and J. Pla. 2009. Msb2 signaling mucin controls activation of Cek1 mitogen-activated protein kinase in Candida albicans. Eukaryot Cell 8:1235-1249. 36. Sabina, J., and V. Brown. 2009. Glucose sensing network in Candida albicans: a sweet spot for fungal morphogenesis. Eukaryot Cell 8:1314-1320. 37. Schaller, M., C. Borelli, H. C. Korting, and B. Hube. 2005. Hydrolytic enzymes as virulence factors of Candida albicans. Mycoses 48:365-377. 38. Sigle, H. C., S. Thewes, M. Niewerth, H. C. Korting, M. Schafer-Korting, and B. Hube. 2005. Oxygen accessibility and iron levels are critical factors for the antifungal action of ciclopirox against Candida albicans. J Antimicrob Chemother 55:663-673. 39. Simonetti, N., V. Strippoli, and A. Cassone. 1974. Yeast-mycelial conversion induced by N-acetyl-D-glucosamine in Candida albicans. Nature 250:344-346. 40. Skrzypek, M. S., M. B. Arnaud, M. C. Costanzo, D. O. Inglis, P. Shah, G. Binkley, S. R. Miyasato, and G. Sherlock. 2010. New tools at the Candida Genome Database: biochemical pathways and full-text literature search. Nucleic Acids Res 38:D428-432.


56

41. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk. 1985. Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85. 42. Sohn, K., C. Urban, H. Brunner, and S. Rupp. 2003. EFG1 is a major regulator of cell wall dynamics in Candida albicans as revealed by DNA microarrays. Mol Microbiol 47:89-102. 43. Sosinska, G. J., P. W. de Groot, M. J. Teixeira de Mattos, H. L. Dekker, C. G. de Koster, K. J. Hellingwerf, and F. M. Klis. 2008. Hypoxic conditions and iron restriction affect the cell-wall proteome of Candida albicans grown under vagina-simulative conditions. Microbiology 154:510-520. 44. Stead, D. A., J. Walker, L. Holcombe, S. R. Gibbs, Z. Yin, L. Selway, G. Butler, A. J. Brown, and K. Haynes. 2009. Impact of the transcriptional regulator, Ace2, on the Candida glabrata secretome. Proteomics. 45. Sudbery, P., N. Gow, and J. Berman. 2004. The distinct morphogenic states of Candida albicans. Trends Microbiol 12:317-324. 46. Swaim, C. L., B. P. Anton, S. S. Sharma, C. H. Taron, and J. S. Benner. 2008. Physical and computational analysis of the yeast Kluyveromyces lactis secreted proteome. Proteomics 8:2714-2723. 47. Thomas, D. P., S. P. Bachmann, and J. L. Lopez-Ribot. 2006. Proteomics for the analysis of the Candida albicans biofilm lifestyle. Proteomics 6:5795-5804. 48. Torosantucci, A., P. Chiani, C. Bromuro, F. De Bernardis, A. S. Palma, Y. Liu, G. Mignogna, B. Maras, M. Colone, A. Stringaro, S. Zamboni, T. Feizi, and A. Cassone. 2009. Protection by anti-beta-glucan antibodies is associated with restricted beta-1,3 glucan binding specificity and inhibition of fungal growth and adherence. PLoS One 4:e5392. 49. Tsuboi, R., K. Matsuda, I. J. Ko, and H. Ogawa. 1989. Correlation between culture medium pH, extracellular proteinase activity, and cell growth of Candida albicans in insoluble stratum corneum-supplemented media. Arch Dermatol Res 281:342-345. 50. Tung, C. W., and S. Y. Ho. 2007. POPI: predicting immunogenicity of MHC class I binding peptides by mining informative physicochemical properties. Bioinformatics 23:942-949. 51. Vadaie, N., H. Dionne, D. S. Akajagbor, S. R. Nickerson, D. J. Krysan, and P. J. Cullen. 2008. Cleavage of the signaling mucin Msb2 by the aspartyl protease Yps1 is required for MAPK activation in yeast. J Cell Biol 181:1073-1081. 52. Whiteway, M., and C. Bachewich. 2007. Morphogenesis in Candida albicans. Annu Rev Microbiol 61:529-553. 53. Williamson, P. R., M. A. Huber, and J. E. Bennett. 1993. Role of maltase in the utilization of sucrose by Candida albicans. Biochem J 291 ( Pt 3):765-771.


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