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Supplemental Table 1. Gluten
immunogenic epitopes (from Sollid
et al., 2012) with highlighted amino
acids preferred by lasB in the P1 (L,
F) and P1’ (G, A) positions.
HLA
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
DQ2.5
epitope
glia-a1a
glia-a1b
glia-a2
glia-a3
glia-g1
glia-g2
glia-g3
glia-g4a
gli-g4b
glia-g4c
glia-g4d
glia-g5
glia-w1
glia-w2
glut-L1
glut-L2
hor-1
hor-2
hor-3
sec-1
sec-2
ave-1a
ave-1b
Structure
PFPQPELPY
PYPQPELPY
PQPELPYPQ
FRPEQPYPQ
PQQSFPEQQ
IQPEQPAQL
QQPEQPYPQ
SQPEQEFPQ
PQPEQEFPQ
QQPEQPFPQ
PQPEQPFCQ
QQPFPEQPQ
PFPQPEQPF
PQPEQPFPW
PFSEQEQPV
FSQQQESPF
PFPQPEQPF
PQPEQPFPQ
PIPEQPQPY
PFPQPEQPF
PQPEQPFPQ
PYPEQEEPF
PYPEQEQPF
DQ2.2
glut-L1
PFSEQEQPV
DQ8
DQ8
DQ8
DQ8
glia-a1
glia-g1a
glia-g1b
glut-H1
EGSFQPSQE
EQPQQPFPQ
EQPQQPYPE
QGYYPTSPQ
DQ8.5
DQ8.5
DQ8.5
glia-a1
glia-g1
glut-H1
EGSFQPSQE
PQQSFPEQE
QGYYPTSPQ
B
A
C
D
1
2 3 4 5 6 7 8 9 10
Supplemental Figure S1. Appearance of some fecal strains on gluten agar (GA) and
their evaluation in a gliadin zymogram. A and B, dilutions of fecal suspensions plated
on GA. C, Colonies showing a clear zone were subcultured to purity on blood agar and
their appearance was confirmed on segmented GA plates. D, evaluation of the eight
strains in C on gliadin zymogram (lanes 2-9). Cells loaded were harvested from 150 µl
OD620 5.0. Left lane: molecular weight standard; right lane: R. mucilaginosa (150 µl
OD620 5.0).
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Supplemental Figure S2. Gliadin zymography of fecal strains. Cells
were harvested from BA and suspended in PBS to an OD620 of 5.0.
Cells in 50 µl aliquots were harvested by centrifugation, resuspended in
zymogram samples buffer and applied to the gel. A, lane 1: MW std;
lanes 2-10: fecal strains FA-28, -30, -31,- 32, -34, -35, -37, -38, -39. B,
lane 1: MW std; lanes 2-9: strains FA-3, -10, -13, -22, -46, -29, -36, -42.
Lane 10: Oral strain R. mucilaginosa ATCC 25296 (50 µl OD620 5.0).
All FA strains were obtained from GA plates at pH 4.0.
Supplemental Figure S3. Selection of fecal strains, grown on gluten
agar for 48 h under aerobic conditions at 37°C. The starting color of the
agar is as shown in the plate on the lower right corner. The other colors
are the result of pigments produced by the bacteria during growth. All
strains were later identified as P. aeruginosa.
M
1
2
3a 3b 4
5
6 7
8
9 10 11 12 13 14 15 16
C M
kDa
250
150
100
75
A
A
50
37
25
20
15
10
M
1
2 3a 3b
4
5
6
7
8
9
10 11 12 13 14 15 16 C
M
B
kDa
250
150
100
75
50
Supplemental Figure S4. SDS-PAGE and casein zymography of P. aeruginosa
proteins separated by DEAE (linear gradient). A, SDS-PAGE (4-12%) of 200 µl
aliquots of the fractions shown in Figure 1B. B, Casein zymogram gel (6%) of
200 μl desalted aliquots of the fractions shown in Figure 1B. M, MW standard;
C: aliquot of the whole P. aeruginosa cell sonicate (pre-DEAE). (5 μl). The SDS
gel was silver-stained, the zymogram gel was stained with Coomassie brilliant
blue. Fractions indicated in red contained the enzyme and eluted as minor peaks
just after the first major peak in the DEAE chromatogram.
EDTA (+)
EDTA (-)
M
F4
F4
M
F4
F4
Supplemental Figure S5. Effect of EDTA on P.
aeruginosa protease activity. Aliquots of 0.04 μg of
isolated protease fraction F4 were subjected to gliadin
zymography (8%) in duplicate under non-reduced
conditions. The gel was divided in two, and one half was
renatured and developed in the absence of EDTA (EDTA), and the other half in the presence of 5 mM EDTA
(EDTA+). Lane 1, MW std; lane 2: empty; lanes 3 and 4:
protease fraction F4 (0.04 µg/lane).
DEAE fractions
with enzyme activity
MW
std
d2 d3 d4 d5 d6 d7 d8 d9 PaE
LC-ESI-MS/MS results:
kDa
250
150
100
75
50
37
25
20
15
10
Commercially
obtained
lasB
1
2
Band 1 (49910):
Total
Unique peptides reference
13
27
G4LSY0_PSEAI
1
1
G4LNV3_PSEAI
Gene Symbol
lasB
cpbD
Band 2 (49911):
Unique
Total
12
36
1
1
1
1
Gene Symbol
lasB
cpbD
fliC
reference
G4LSY0_PSEAI
G4LNV3_PSEAI
G4LR53_PSEAI
Supplemental Figure S6. Isolation and identification of the lasB enzyme from P. aeruginosa
strain FA-10. A, SDS PAGE; B, LC-ESI-MS/MS results of two bands excised from the SDS
PAGE gel (bands in fractions d5 and d6). The protein migrated at the expected molecular
weight in the gel and was conclusively identified as lasB.
Supplemental Figure S7. X-ray crystalographic resolution of
pseudolysin at 1.5Å. The catalytic zinc is shown as a light grey
CPK sphere. The structural calcium ion is shown as a yellow CPK
sphere. The zinc ligands are shown in ball-and-stick representation:
His337 and His341 in purple and Glu361 in blue. Catalytic residues
are shown in ball-and-stick representation: Glu141 in blue, Asp168
and Asp338 in pink, and His420 in purple. (From:
http://merops.sanger.ac.uk).
-/-/-/g ┼ Lfiv/a/-/- (based on 70 cleavages)
Amino acid
P4
P3
P2
P1
P1'
P2'
P3'
P4'
Gly
3
8
7
26
0
8
10
6
Pro
2
0
2
3
0
0
5
6
Ala
3
6
6
8
4
21
2
2
Val
2
3
3
1
6
5
1
1
Leu
3
4
1
1
35
2
1
0
Ile
3
1
1
0
6
3
1
1
Met
0
0
0
1
1
0
2
0
Phe
1
5
5
7
12
2
1
1
Tyr
1
1
1
2
4
2
3
0
Trp
0
0
0
0
1
0
0
0
Ser
3
2
3
5
0
3
2
3
Thr
2
1
0
2
0
6
1
3
Cys
0
1
0
0
0
0
0
0
Asn
0
1
1
2
0
2
0
1
Gln
1
1
5
1
0
1
1
1
Asp
1
1
0
1
0
0
2
1
Glu
4
2
2
1
0
2
3
4
Lys
2
2
4
4
0
2
1
3
Arg
5
2
7
2
0
2
3
3
His
0
0
0
3
0
0
0 0
Supplemental Figure S8. Cleavage specificity information of
pseudolysin from P. aeruginosa. (From: http://merops.sanger.ac.uk).