Linking Fungal Virulence and Secretome: Comprehensive Analysis
of Proteins Secreted by Trichophyton rubrum and T. violaceum
Karin Giddey1, Michel Monod1, Jachen Barblan2, Alexandra Potts2, Patrice Waridel2,
Christophe Zaugg1 and Manfredo Quadroni2
1
2
Service de Dermatologie et Venereologie Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland,
Protein Analysis Facility, Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland
Introduction
Methods
Dermatophytes are highly specialized pathogenic fungi which cause most
superficial mycoses in humans and animals [1]. Among the approximately 10
human pathogenic species isolated in Europe, Trichophyton rubrum is the most
commonly observed. T. violaceum, which is mainly responsible for infection of
the scalp in North African, Middle East and Mediterranean countries, is closely
related to T. rubrum. The pathogenic specificity of these fungi is probably linked
to the secretion of proteins degrading keratinised structures in the stratum
corneum. In the present study [2], we investigated the major secreted proteins
from T. rubrum (2 strains: 250 & 1738) ) and T. violaceum (819) under in vitro
conditions which promote protein secretion and to some extent mimic in vivo
growth parameters.
The lack of complete dermatophyte genome sequence information has forced us to exert special care in data
evaluation. We have thus employed two independent techniques, 2D-PAGE and a shotgun mass spectrometry
approach, to cross-validate the results.
>2D-PAGE: spot detection and matching with PDQuest 4.0 (Bio-RAD), MALDI TOF MS/MS analyses of digested
spots with 4700 Proteomics Analyzer (Applied Biosystems), LC-MS/MS analyses with SCIEX QSTAR Pulsar I
(Applied Biosystems)
>1D-PAGE shotgun: limited electrophoretic separation (about 2.5cm) on 10% gels, lanes cut in 9 bands, LCMS/MS analyses of digested bands with SCIEX QSTAR Pulsar I (Applied Biosystems), 2 injections/sample (one
maximizing sensitivity, the second maximizing number of precursors), data pooled (about 20’000 MS/MS
spectra/strain), relative quantification by spectral counting [4] (percentage of matched spectra)
> Database search: MASCOT search with T. rubrum composite protein database (9280 EST-derived amino acid
sequences (TrED, [3]) + 65 Uniprot sequences), identifications validated with Scaffold (Proteome Software).
Results
¾ 2D-PAGE of both species showed a similar pattern and spots matched were assigned to the same databases sequences with similar coverage, showing that T. rubrum
database could be used for T violaceum proteomic analyses. Some protein degradation was observed but remained limited (Figure 1).
¾ Considering both species together we were able to identify 80 secreted proteins in shotgun analyses. Identified proteins included numerous endo- and exo- proteases, other
hydrolases, oxidoreductases as well as proteins of unknown function (Figure 2), Based on spectral counting, proteases represented the major part of secreted proteins (Figure
3). Comparison of secreted proteins from T. rubrum and T. violaceum revealed a high global level of similarity, but also significant differences. For example, often a protease
detected in T. rubrum was substituted by another member of the same family in T. violaceum (Figure 4):
Mep3 → Mep2, Sub3 → Sub6,
DppIV → DppIV/DppV.
Figure 3: Relative amount of secreted proteins
Figure 1: 2D PAGE
3.0
10.0
T. rubrum 250
1
2
4
3
8
50kD
1%
T. violaceum
10
13
12
14
1%
13%
17
15
4%
16
19
7%
Proteases
Other hydrolases
Other enzymes
Non enzymes
Unknown function
22%
20
22
21
25kD
26
15kD
31
29
28
24
23
25
20kD
10%
35
37
36
33
Figure 4: Relative amount of secreted proteases
10kD
3.0
10.0
T. violaceum 819
T. rubrum 250
1
2
5
10
22
21
24
23
25
20kD
26
15kD
31
27
34
37
29
30
12.00
33
35
36
10kD
8.00
6.00
4.00
2.00
Transferases
Non-enzymes
Unknown function
Proteases (29 %)
S03304
Sub7
Sub6
Sub4
Sub3
Mep4
Mep3
S14420
Oxidoreductases
Unknown function
(17%)
S08469
Isomerases
CPY
Others
S01160
Lipases
Non-enzymes (10%)
S00686
Glycosidases
PAP
Exoproteases
6.00
5.00
4.00
3.00
2.00
1.00
0.00
DppV
Endoproteases
26.00
23.00
20.00
7.00
Lap1
Other enzymes (19 %)
B)
% matched spectra
Figure 2: Secreted proteins identified in
T. rubrum and T. violaceum (all strains)
Mep1
0.00
Two-dimensional separation of proteins secreted by T. rubrum LAU250 and T. violaceum
LAU819. Staining was by Coomassie Brilliant Blue. Boxes group clusters of spots assumed to
be glycoforms of the same protein by electrophoretic pattern and / or identification by mass
spectrometry ( proteases, other hydrolases, other function, not identified).
Other hydrolases (25 %)
10.00
S11873 (DppIV)
28
32
T. violaceum 819
16
20
25kD
T. rubrum 1738
14.00
DppIV
19
17
15
14
18
13
12
11
37kD
A)
7
9
8
50kD
6
% matched spectra
4
3
Mep2
250kD
150kD
100kD
75kD
Lap2
pI
59%
27
34
32
30
58%
24%
S11391
18
7
S12285
11
37kD
T. rubrum
6
5
9
S14596
pI
250kD
150kD
100kD
75kD
Spectral counts for proteases detected by the shotgun technique (percentages of matched spectra normalised
for every strain). Panel A), endoproteases, B), exoproteases. In A) from left to right fungalysins (Mep1 to
Mep4), subtilisins (Sub3 to Sub7), uncharacterized ones. In B) from left to right leucine aminopeptidases
(Lap1, Lap2), dipeptyl-peptidases (DppIV, DppV), putative prolyl aminopeptidases (Q52H54,S00686), putative
aspartyl aminopeptidase (S01160), putative carboxypeptidases (CPY to S14420).
Conclusions
References
The endo- and exoproteases which were identified in this study constitute a
machinery capable of digesting an intact polypeptidic chain into amino acids and
short peptides. During this process of degradation, the action of endoproteases
generates a large number of smaller peptides, which in turn are attacked by the
exoproteases. Our findings suggest that the difference in habitat and phenotype
between both species could be related to a different pattern of secreted proteins.
1.
2.
3.
4.
Weitzman, I., Summerbell, R.C. Clin. Microbiol. Rev. 1995, 8(2), 240-259.
Giddey, K., Monod, M., Barblan, J. et al. J. Proteome Res. 2007, 6 (8), 3081-3092.
Wang, L., Ma., L., Leng, W. et al. BMC Genomics 2006, 7, 255.
Liu, H.; Sadygov, R. G.; Yates, J. R., 3rd. Anal Chem 2004, 76 (14), 4193-4201.