9. Appendix
9.1. List of abbreviations
2D DIGE 2 dimensional difference gel electrophoresis 2D GE 2 dimensional gel electrophoresis
AIDS Acquired Immune Deficiency Syndrome
bp Base pair(s)
β Beta
BSA Bovine Serum Albumin
CO2 Carbon dioxide
cm Centimetre
COG Cluster of Orthologous Group CD Crohn's disease
ESI Q-TOF Electrospray ionization quadrupole TOF et al. Et alii
etc. Et cetera e.g. Exampli gratia Fig. Figure
GeLC-MS Gel-based liquid chromatography-mass spectrometry
g Gram(s)
g Gravitational constant HEYM Herrold’s egg yolk medium h Hour(s)
Appendix MLSSR Multilocus short sequence repeat
MIRU-VNTR Multiple-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat
M. Mycobacterium MAC Mycobacterium avium complex MAA Mycobacterium avium subsp. avium MAH Mycobacterium avium subsp. hominissuis MAP Mycobacterium avium subsp. paratuberculosis MAP Mycobacterium avium subsp. paratuberculosis
n Nano
ng Nano gram(s)
nUPLC Nano ultra performance liquid chromatography nm Nanometer
OADC Oleic Acid Albumin Dextrose Complex ORF Open Reading Frame PBS Phosphate Buffered Saline
PAGE Polyacrylamide gel electrophoresis PCR Polymerase chain reaction
pH Power of Hydrogen
PLP pyridoxal-5’-phosphate NOS Reactive nitrogen species ROS Reactive oxygen species
RCN Reference common name
® Registered trademark
RFLP Restriction length polymorphism RT-PCR Reverse Transcriptase PCR
RNA RiboNucleic Acid rpm Rounds per minute s Second(s)
SDS Sodium Dodecyl Sulphate MS/MS Tandem mass spectrometry TOF Time-of-flight
TM Trade mark
TCA Tricarboxylic acid cycle
Tris Tris-(hydroxymethyl-) aminomethane
U Unit
VBNC Viable but non culturable V Volt
Vol Volume v/v Volume by volume
WR Watson Reid
w/v Weight by volume
Appendix
146 9.2. Complete results of 2D GE/2D DIGE:
The following figures illustrate all results from comparative and preparative 2D gelelectrophoresis (2D DIGE and 2D GE) with cytoplasmic fractions of in vivo and in vitro grown MAP with protein identifications, visualized by arrows and spot numbers, were listed in the respective Supplementary Tables S 9-1 – 9-7. In the 2D DIGE pictures, proteins from CDC were labelled with Cy 3 are green and proteins from MDC were labelled with Cy 5 are red. If proteins from CDC and MDC were similar expressed, they were represented in yellow spots in 2D DIGE overlay pictures. All together, 331 spots were excised from seven preparative 2D gels and subsequently identified using MALDI-TOF-MS revealing 153 protein identifications specific for MAP. Figures 1, 4 and 6 demonstrates Coomassie stained preparative 2D gels with MDC from cow 2 (a) and 2D DIGE gel with CDC from cow 2 (green) and MDC from cow 2 (red) and figures 2, 3, 5 and 7 with samples prepared from cow 3 respectively.
,
Supplementary Fig. S 9-1. Protein spots from 2D GE with MDC (1a) and 2D DIGE with MDC vs. CDC (1b) from cow 2 (batch 1).
Appendix
148
Supplementary Table S 9-1. Identified proteins excised from corresponding regions at Coomassie stained preparative 2D gel with MDC from cow2.
Respective 2D gel picture and corresponding 2D DIGE gel picture are shown in Supplementary Fig 1a and Supplementary Fig. 1b.
change Putative function RCN COG
39 44.3 4.9 87 9 MAP2451c Rv1310 / F0F1 ATP synthase subunit beta AtpD C 42 43.3 4.9 244 19 MAP1126 Rv1392 2.68 S-adenosylmethionine synthetase MetK H 43 43.3 4.9 74 6 MAP1126 Rv1392 / S-adenosylmethionine synthetase MetK H
62 92.6 5.2 193 23 MAP3853 Rv0384c 1.49 ATP-dependent chaperone ClpB ClpB O 64 81.8 5.0 100 11 MAP1668c Rv1908c 2.27 Catalase/peroxidase KatG P 65 78.7 5.1 176 12 MAP3716c Rv0271c 2.32 Putative acyl-CoA dehydrogenase FadE6 I 66 78.7 5.1 206 16 MAP3716c Rv0271c 2.62 Putative acyl-CoA dehydrogenase FadE6 I 67 60.2 4.9 82 9 MAP2453c Rv1308 4.18 F0F1 ATP synthase subunit alpha AtpA C 70 39.9 5.2 78 8 MAP2646c 1.17 Acetyl-CoA acyltransferase FadA6_3 I 71 36.2 5.2 137 10 MAP1164 Rv1436 -1.16 Glyceraldehyde-3-phosphate
dehydrogenase Gap G
72 33.4 5.3 168 13 MAP3190 Rv3141 1.24 Putative NADPH2:quinone
reductase FadB4 CR
75 31.3 5.2 188 14 MAP0907 1.41 Putative aldo/keto reductase
family R
The Functional classification of proteins identified has been adopted from the genome database of M. avium subsp. paratuberculosis K-10 (http://www.ncbi.nlm.nih.gov/sutils/coxik.cgi?gi=380) according to the database Cluster of Orthologous Groups of Proteins (COGs). ‡ Protein scores greater than 73 are significant (p<0.05).
Supplementary Fig. S 9-2. Protein spots from 2D GE with MDC (2a) and 2D DIGE with MDC vs. CDC (2b) from cow 3 (batch 1).
Appendix
150
Supplementary Table S 9-2. Identified proteins excised from corresponding regions at Coomassie stained preparative 2D gel with MDC from cow3.
Respective 2D gel picture and corresponding 2D DIGE gel picture are shown in Supplementary Fig. 2a and Supplementary Fig. 2b.
change Putative function RCN COG
1 92.6 5.2 116 15 MAP3853 Rv0384c / ATP-dependent chaperone ClpB O
5 54.6 5.0 178 18 MAP3362c Rv3248c 1.32 S-adenosyl-L-homocysteine
hydrolase SahH H
7 44.3 6.2 108 16 MAP3651c Rv0215c 1.27 Putative acyl-CoA dehydrogenase FadE3_2 I 8 42.7 5.0 121 9 MAP0789 Rv0859 1.61 Acetyl-CoA acetyltransferase FadA_1 I 9 40.7 5.9 189 14 MAP1205 Rv1479 1.12 MoxR-like ATPase MoxR R 10 41.7 5.7 188 15 MAP0244 Rv3778c 1.41 Putative Selenocysteine
lyase/Cysteine desulfurase E 10 40.7 5.9 77 7 MAP1205 Rv1479 1.41 MoxR-like ATPase MoxR R 11 41.7 5.7 161 15 MAP0244 Rv3778c 1.73 Putative Selenocysteine
lyase/Cysteine desulfurase E 11 40.7 5.9 73 8 MAP1205 Rv1479 1.73 MoxR-like ATPase MoxR R 13 55.8 4.9 88 9 MAP4265 Rv3417c 21.03 Chaperonin GroEL1 GroEL1 O 14 33.4 5.3 153 11 MAP3190 Rv3141 1.11 Putative NADPH2:quinone
reductase FadB4 CR
15 35.1 5.4 88 6 MAP1613c / Alcohol dehydrogenase AdhA_2 R 16 35.4 5.9 148 13 MAP1361 Rv1652 1.5
N-acetyl-gamma-glutamyl-phosphate reductase ArgC E 17 33.3 4.6 77 7 MAP3152c Rv3075c 1.4 Putative malate synthase G 18 32.1 4.7 84 7 MAP3060c 1.3 Putative electron transfer
flavoprotein alpha subunit FixB C 19 27.8 4.9 89 9 MAP3061c Rv3029c 1.29 Putative electron transfer
flavoprotein beta subunit FixA C 20 33.3 5.1 125 10 MAP4095c Rv0644c 1.35
Cyclopropane-fatty-acyl-phospholipid synthase MmaA2 M 20 33.6 5.3 73 10 MAP4308c 1.35 Fructose-1,6-bisphosphate aldolase G 21 40.7 5.9 81 8 MAP1205 Rv1479 1.51 MoxR-like ATPase MoxR R 33 11.5 5.4 68 † 3 MAP0467c Rv3592 1.62 Putative antibiotic biosynthesis
monooxygenase R
For further information see legend for Supplementary Table S 9-1. † Protein has given a significant hit excised at similar positions from other 2D gels.
Supplementary Fig. S 9-3. Protein spots from 2D GE with MDC (3a) and 2D DIGE with MDC vs. CDC (3b) from cow 3 (technical repeat batch 1).
Appendix
152
Supplementary Fig S 9-3 cont. (for figure legends see page 151).
Supplementary Table S 9-3. Identified proteins excised from corresponding regions at Coomassie stained preparative 2D gel with MDC from cow3 (technical repeat of 2D GE/2D DIGE from Fig. 2a, b and Table 2).
Respective 2D gel picture and corresponding 2D DIGE gel picture are shown in Fig. 3a and Fig. 3b.
Spot
change Putative function RCN COG
5 101.8 4.8 99 10 MAP1201c Rv1475c 2.1 Aconitate hydratase Acn C 13 54.6 5.0 152 11 MAP3362c Rv3248c 1.4 S-adenosyl-L-homocysteine
hydrolase SahH H 17 42.0 5.2 75 9 MAP3392c Rv3274c / Acyl-CoA dehydrogenase FadE25_4 I 20 40.2 5.1 281 11 * MAP0595c Rv0761c 2.16
S-(hydroxymethyl)glutathione dehydrogenase / alcohol dehydrogenase
AdhB C 21 42.1 5.4 153 12 MAP3877c 4.07 Acyl-CoA dehydrogenase FadE20_3 I 21 39.4 5.5 103 8 MAP2276c Rv2454c 4.07 2-oxoglutarate ferredoxin
oxidoreductase subunit beta C 22 33.7 5.0 124 7 MAP2121c 1.12 Major membrane protein I (-) 23 36.1 5.0 91 7 MAP3668c Rv0230c 3.8 Putative
phosphotriesterase-related protein R
23 55.8 4.9 81 6 MAP4265 Rv3417c 3.8 Chaperonin GroEL1 GroEL1 O 24 39.4 5.0 101 9 MAP2228 3.8 Putative acyl-CoA
dehydrogenase I
26 33.6 4.9 52 † 4 MAP1365 Rv1656 2.04 Ornithine carbamoyltransferase ArgF E 27 35.4 5.9 154 10 MAP1361 Rv1652 1.18
N-acetyl-gamma-glutamyl-phosphate reductase ArgC E 28 32.3 4.9 93 7 MAP2123 Rv2334 3.09 Cysteine synthase CysK E 29 24.1 5.0 48 † 4 MAP4196 Rv0730 / Putative acetyltransferase
(GNAT) family L
30 30.8 5.2 96 11 MAP0897 Rv0952 1.26 Succinyl-CoA synthetase subunit
alpha SucD C
31 24.9 4.8 357 14 * MAP3983 Rv0491 1.64 Response regulator RegX3 RegX3 TK 32 31.5 4.9 159 9 MAP2698c Rv1094 / Acyl-[acyl-carrier-protein]
desaturase DesA2 (-)
34 26.2 5.3 124 9 MAP3116c 2.05 Putative short chain
dehydrogenase IQR
35 23.3 5.0 81 5 MAP2280c Rv2460c -1.37 ATP-dependent Clp protease
proteolytic subunit ClpP2 OU 37 20.6 5.4 55 † 3 MAP2945c Rv2882c / Ribosome recycling factor Frr J 39 19.0 5.0 44 † 2 MAP1588c Rv2429 / Alkyl hydroperoxide reductase
subunit D AhpD S
41 15.5 4.8 81 5 MAP2411 / Putative pyridoxamine
5'-phosphate oxidase (-)
Appendix
154
Supplementary Table S 9-3 cont.
Spot
# MW
[Da] pI Score
‡
Matched
peptides Locus tag H37Rv Fold
change Putative function RCN COG
43 11.5 5.4 207 4 * MAP0467c Rv3592 1.64 Putative antibiotic biosynthesis
monooxygenase R
44 34.7 4.9 112 9 MAP2541c Rv1240 1.16 Malate dehydrogenase Mdh C 45 32.6 5.0 86 7 MAP1175c Rv1446c 2 Putative glucose-6-phosphate
dehydrogenase OpcA G
46 53.8 4.6 200 19 MAP3659 2.8 Acyl-protein synthetase (LuxE) IQ 48 49.9 5.8 169 12 * MAP1587c / Putative alpha-amylase (AmyA) G 49 24.0 5.0 255 6 * MAP0268c 1.51 Putative SAM dependent
methyltransferase (-) For further information see legend for Supplementary Table S 9-1. † Protein has given a significant hit excised at similar positions from other 2D gels. * Peptides have been matched by MALDI-TOF/TOF.
Supplementary Fig. S 9-4. Protein spots from 2D GE with MDC (4a) and 2D DIGE with MDC vs. CDC (4b) from cow 2 (batch 2).
Supplementary Fig S 9-4 cont. (for figure legends see page 154).
Appendix
156
Supplementary Table S 9-4. Identified proteins excised from corresponding regions at Coomassie stained preparative 2D gel with MDC from cow2.
Respective 2D gel picture and corresponding 2D DIGE gel picture are shown in Fig. 4a and Fig. 4b.
Spot
change Putative function RCN COG
1 109.7 5.2 collagen alpha-1(VI) chain [Bos
12 81.7 5.0 139 8 MAP1668c Rv1908c 6 Catalase/peroxidase KatG P 13 81.7 5.0 98 6 MAP1668c Rv1908c 12.48 Catalase/peroxidase KatG P 33 66.6 5.0 89 6 MAP3694c Rv0244c 3.46 Putative acyl-CoA
dehydrogenase FadE5 I 34 60.2 4.9 78 5 MAP2453c Rv1308 12.33 F0F1 ATP synthase subunit
alpha AtpA C
35 60.2 4.9 79 5 MAP2453c Rv1308 10.66 F0F1 ATP synthase subunit
alpha AtpA C
52 49.7 5.1 96 5 MAP1368 Rv1659 2 Argininosuccinate lyase ArgH E 54 49.2 5.1 82 5 MAP1024 Rv1077 1.2 Cysteine synthase CysM2 E 56 43.2 4.9 189 11 MAP1126 Rv1392 5.9 S-adenosylmethionine synthetase MetK H 68 37.7 4.7 224 17 * MAP4233 Rv3457c / DNA-directed RNA polymerase
subunit alpha RpoA K
69 40.4 4.7 239 15 MAP1177c Rv1448c 2.69 Transaldolase Tal G 71 66.5 4.7 199 17 * MAP3840 Rv0350 7.41 Molecular chaperone DnaK O 74 39.9 4.8 109 10 MAP0823c Rv0884c 2 Phosphoserine aminotransferase SerC HE 80 42.0 5.2 66 † 11 * MAP3392c Rv3274c 4.39 Acyl-CoA dehydrogenase FadE25_4 I 88 33.4 5.0 110 7 MAP1563c Rv1855c / Putative F420-dependent
oxidoreductase C
90 36.1 5.0 108 5 * MAP3668c Rv0230c 2.33 Putative
phosphotriesterase-related protein R 138 37.7 4.7 78 4 MAP4233 Rv3457c 4.38 DNA-directed RNA polymerase
subunit alpha RpoA K
149 27.8 4.7 100 8 MAP3061c Rv3029c 1.37 Putative electron transfer
flavoprotein beta subunit FixA C 163 30.3 6.3 99 6 MAP4227c Rv3463 3.56 Putative luciferase-like
monooxygenase C
182 20.6 5.4 98 5 MAP2945c Rv2882c / Ribosome recycling factor Frr J For further information see legend for Supplementary Table S 9-1. † Protein has given a significant hit excised at similar positions from other 2D gels. * Peptides have been matched by MALDI-TOF/TOF.
Supplementary Fig. S 9-5. Protein spots from 2D GE with MDC (5a) and 2D DIGE with MDC vs. CDC (5b) from cow 3 (batch 2).
Appendix
158
Supplementary Fig S 9-5 cont. (for figure legends see page 157).
Supplementary Table S 9-5. Identified proteins excised from corresponding regions at Coomassie stained preparative 2D gel with MDC from cow3.
Respective 2D gel picture and corresponding 2D DIGE gel picture are shown in Fig. 5a and Fig. 5b.
Spot
change Putative function RCN COG
1 109.7 5.2 collagen alpha-1(VI) chain [Bos 13 54.4 5.0 157 13 MAP3362c Rv3248c 1.21 S-adenosyl-L-homocysteine
hydrolase SahH H
14 56.6 4.9 390 16 * MAP3936 Rv0440 4.76 Chaperonin GroEL GroEL2 O 17 43.7 5.2 122 7 MAP1367 Rv1658 1.81 Argininosuccinate synthase ArgG E 18 43.7 5.2 172 11 MAP1367 Rv1658 5.5 Argininosuccinate synthase ArgG E 21 39.4 5.5 95 9 MAP2276c Rv2454c 2 2-oxoglutarate ferredoxin
oxidoreductase subunit beta C 23 55.8 4.9 124 9 MAP4265 Rv3417c 6.1 Chaperonin GroEL1 GroEL1 O 26 33.6 4.9 87 5 MAP1365 Rv1656 / Ornithine carbamoyltransferase ArgF E 27 35.4 5.9 179 11 MAP1361 Rv1652 1.17
N-acetyl-gamma-glutamyl-phosphate reductase ArgC E 28 32.3 4.9 407 9 * MAP2123 Rv2334 2.7 Cysteine synthase CysK E 29 24.1 5.0 116 8 * MAP4196 Rv0730 2.09 Putative acetyltransferase
(GNAT) family L
Q-TOF) MAP2058c 2.2 3'(2'), 5'-bisphosphate
nucleotidase CysQ_2 P
MAP1588c Rv2429 / Alkyl hydroperoxide reductase
subunit D AhpD S
41 15.5 4.8 97 6 MAP2411 3.04 Putative pyridoxamine
5'-phosphate oxidase (-)
44 34.7 4.9 79 6 MAP2541c Rv1240 1.01 Malate dehydrogenase Mdh C
Appendix
160
For further information see legend for Supplementary Table S 9-1. † Protein has given a significant hit excised at similar positions from other 2D gels. * Peptides have been matched by MALDI-TOF/TOF.
Supplementary Fig. S 9-6. Protein spots from 2D GE with MDC (6a) and 2D DIGE with MDC vs. CDC (6b) from cow 2 (batch 3).
Supplementary Fig S 9-6 cont. (for figure legends see page 160).
Appendix
162
Supplementary Table S 9-6. Identified proteins excised from corresponding regions at Coomassie stained preparative 2D gel with MDC from cow2.
Respective 2D gel picture and corresponding 2D DIGE gel picture are shown in Fig. 6a and Fig. 6b.
Spot
change Putative function RCN COG
2 109.7 5.2 75 8 collagen alpha-1(VI) chain [Bos 22 75.7 4.9 117 10 MAP3951c Rv0457c 8.03 Uncharacterised protein
23 81.8 5.0 95 9 MAP1668c Rv1908c 10.13 Catalase/peroxidase KatG P
24 81.8 5.0 216 17 MAP2661 Rv1133c 2.15 5-methyltetrahydropteroyltri-glutamate-homocysteine 30 78.7 5.1 103 8 MAP3716c Rv0271c 2.27 Putative acyl-CoA dehydrogenase FadE6 I 31 78.7 5.1 200 16 MAP3716c Rv0271c 2.02 Putative acyl-CoA dehydrogenase FadE6 I 32 60.2 4.9 89 7 * MAP2453c Rv1308 6.18 F0F1 ATP synthase subunit alpha AtpA C 37 66.6 5.0 172 11 * MAP3694c Rv0244c 3.86 Putative acyl-CoA dehydrogenase FadE5 I 38 59.9 5.7 200 14 MAP2863c Rv2752c 1.67 Putative metallo-beta-lactamase R 39 59.1 5.8 259 19 MAP2593c Rv1187 3.63 1-pyrroline-5-carboxylate
dehydrogenase RocA C
53 43.3 4.9 191 12 MAP1126 Rv1392 3.14 S-adenosylmethionine synthetase MetK H 56 40.2 5.0 120 8 MAP2436c Rv1323 2.23 Acetyl-CoA acetyltransferase FadA4 I 57 47.9 5.0 115 6 * MAP3861 2.11 3-oxoacid CoA-transferase ScoB I 58 48.3 5.1 201 16 MAP0829 Rv0896 1.3 Type II citrate synthase GltA2 C 59 48.2 5.1 153 16 * MAP0829 Rv0896 1.79 Type II citrate synthase GltA2 C
Supplementary Table S 9-6 cont.
change Putative function RCN COG
59 42.4 5.2 175 11 * MAP3871 1.79 Phosphoribosylglycinamide
formyltransferase 2 PurT F 60 48.3 5.1 162 13 MAP0829 Rv0896 1.35 Type II citrate synthase GltA2 C 61 43.7 5.2 128 10 MAP4143 Rv0685 -2.2 Elongation factor Tu Tuf J 62 43.9 5.2 173 14 MAP1367 Rv1658 1.02 Argininosuccinate synthase ArgG E 63 45.0 5.8 180 16 MAP2699c Rv1093 1.35 Serine hydroxymethyltransferase GlyA E 64 46.6 5.6 236 19 * MAP3455c Rv3339c -1.16 Isocitrate dehydrogenase 1 Icd1 C 65 45.0 5.8 124 10 MAP2699c Rv1093 / Serine hydroxymethyltransferase GlyA E 66 44.9 5.7 127 10 MAP3334 Rv3227 -3.6 3-phosphoshikimate
1-carboxyvinyltransferase (AroA) AroA E 66 43.1 5.5 87 8 MAP3680c -3.6 Formate dehydrogenase CHR 67 49.9 5.8 154 10 MAP1587c / Putative alpha-amylase (AmyA) G 68 49.9 5.8 132 11 MAP1587c / Putative alpha-amylase (AmyA) G 69 39.9 4.8 75 8 MAP0823c Rv0884c 1.24 Phosphoserine aminotransferase SerC HE 70 42.5 4.8 146 13 MAP1165 1.85 Phosphoglycerate kinase Pgk G 71 43.2 5.0 33 † 2 MAP0896 Rv0951 1.72 Succinyl-CoA synthetase subunit
beta SucC C
72 33.4 5.0 96 7 MAP1563c Rv1855c 2.54 Uncharacterised protein C 74 40.2 5.1 113 7 * MAP0595c Rv0761c 2.97 S-(hydroxymethyl)glutathione
dehydrogenase / alcohol dehydrogenase
AdhB C 75 42.0 5.2 94 7 * MAP3392c Rv3274c 4.57 Acyl-CoA dehydrogenase FadE25_4 I 76 42.0 5.2 95 8 * MAP3392c Rv3274c / Acyl-CoA dehydrogenase FadE25_4 I 78 45.2 5.3 106 9 MAP1052 Rv2580c 1.81 Histidyl-tRNA synthetase HisS J 79 39.4 5.5 92 8 MAP2276c Rv2454c 3.55 2-oxoglutarate ferredoxin
oxidoreductase subunit beta C 80 42.3 5.6 215 17 MAP0827c Rv0889c 1.34 Citrate synthase CitA C 82 41.7 5.7 174 13 MAP0244 Rv3778c 1.79 Putative Selenocysteine
lyase/Cysteine desulfurase E 83 35.4 5.9 142 12 MAP1361 Rv1652 1.46
N-acetyl-gamma-glutamyl-phosphate reductase ArgC E 84 39.3 5.9 104 9 MAP0658c Rv0824c / Acyl-[acyl-carrier-protein]
desaturase DesA1 (-)
85 38.1 6.1 113 10 MAP2909c Rv2841c 1.01 Transcription elongation factor NusA K 85 37.3 6.1 75 7 MAP3044c Rv3010c 1.01 6-phosphofructokinase (PfkA) PfkA G 86 34.7 4.9 81 7 MAP2541c Rv1240 / Malate dehydrogenase Mdh C 86 33.7 4.9 48 † 4 MAP1365 Rv1656 / Ornithine carbamoyltransferase ArgF E 88 36.1 5.0 77 8 MAP3668c Rv0230c 2.01 Putative phosphotriesterase-related
protein R
90 25.3 4.7 36 6 microfibril-associated glycoprotein 4
[Bos taurus].
92 34.2 5.3 129 8 MAP1180c -1.23 Quinone oxidoreductase Qor CR 94 32.7 5.9 138 9 MAP0494 -2.49 Putative NAD dependent
epimerase/dehydratase family MG 95 33.5 5.2 102 8 MAP3402 Rv3283 -1.01 Putative rhodanese-related
sulfurtransferase SseA P 98 28.7 4.7 144 12 * MAP0630c Rv0798c 2.02 Putative encapsulating protein for
peroxidase S
Appendix
change Putative function RCN COG
99 27.8 4.7 106 8 MAP3061c Rv3029c 1.81 Putative electron transfer
flavoprotein beta subunit FixA C 100 31.6 4.9 126 10 MAP2698c Rv1094 2.55 Acyl-[acyl-carrier-protein]
desaturase DesA2 (-)
103 24.1 5.0 181 8 * MAP4196 Rv0730 2.64 Putative acetyltransferase (GNAT)
family L
105 22.9 5.1 98 4 * MAP0622c Rv0786c / Putative metallo-beta-lactamase
(MAV_0733) ‡ R
108 27.3 5.4 143 10 MAP3981 Rv0489 -1.04 Phosphoglyceromutase Gpm G 109 27.9 5.5 254 18 * MAP1017c Rv1070c 1.77 Enoyl-CoA hydratase EchA8_1 I 111 30.2 5.7 133 7 MAP3567 Rv0148 1.08 Putative short chain dehydrogenase IQR 112 31.4 5.7 126 7 MAP2709c Rv2605c -1.22 Acyl-CoA thioesterase II TesB2 I 114 24.2 6.0 74 5 MAP3475c Rv3368c -1.05 Putative Nitroreductase C 115 30.3 6.3 148 10 MAP4227c Rv3463 1.89 Putative luciferase-like
monooxygenase C
93a 33.6 5.3 112 14 MAP4308c 1.4 Fructose-1,6-bisphosphate aldolase G 93b 33.3 5.1 168 12 MAP4095c Rv0644c 1.4
Cyclopropane-fatty-acyl-phospholipid synthase MmaA2 M For further information see legend for Supplementary Table S 9-1. † Protein has given a significant hit excised at similar positions from other 2D gels. * Peptides have been matched by MALDI-TOF/TOF.
Supplementary Fig. S 9-7. Protein spots from 2D GE with MDC (7a) and 2D DIGE with MDC vs. CDC (7b) from cow 3 (batch 3).
Appendix
166
Supplementary Fig S 9-7 cont. (for figure legends see page 165).
Supplementary Table S 9-7. Identified proteins excised from corresponding regions at Coomassie stained preparative 2D gel with MDC from cow3.
Respective 2D gel picture and corresponding 2D DIGE gel picture are shown in Fig. 7a and Fig. 7b.
Spot
change Putative function RCN COG
1 79.9 4.9 83 5 MAP3262c 1.53 Glycogen operon protein GlgX_2 G
2 81.8 5.0 154 17 MAP1668c Rv1908c 3.95 Catalase/peroxidase KatG P 3 81.8 5.0 162 15 MAP1668c Rv1908c 7.32 Catalase/peroxidase KatG P 4 99.2 5.1 91 10 MAP2271c Rv2448c -2.26 Valyl-tRNA synthetase ValS J 14 78.7 5.1 207 15 MAP3716c Rv0271c / Putative acyl-CoA
dehydrogenase FadE6 I 15 76.7 5.2 76 6 MAP3115c 1.62 Putative acyl-CoA
dehydrogenase FadE22 I 16 71.9 5.2 130 11 MAP2101 1.98 Putative acyl-CoA oxidase I 17 68.1 5.3 389 25 MAP0262 Rv3762c -1.24 Metallo-beta-lactamase
superfamily Q
19 70.9 5.8 155 11 MAP3698c Rv0248c 2.73 Succinate dehydrogenase
flavoprotein subunit SdhA C 20 66.5 4.7 226 15 MAP3840 Rv0350 1.36 Molecular chaperone DnaK O 21 60.2 4.9 174 15 * MAP2453c Rv1308 7.65 F0F1 ATP synthase subunit alpha AtpA C 22 60.2 4.9 103 7 MAP2453c Rv1308 16.09 F0F1 ATP synthase subunit alpha AtpA C 23 59.4 5.1 144 9 MAP3399 Rv3280 1.83 Methylmalonyl-CoA
carboxyltransferase AccD5 I 24 63.6 5.2 175 12 MAP2913c Rv2845c 1.09 Prolyl-tRNA synthetase ProS J 25 59.1 5.8 158 11 MAP2593c Rv1187 / 1-pyrroline-5-carboxylate
dehydrogenase RocA C
26 59.1 5.8 170 11 MAP2593c Rv1187 3.39 1-pyrroline-5-carboxylate
dehydrogenase RocA C
28 66.6 5.0 82 6 MAP3694c Rv0244c 3.78 Putative acyl-CoA
dehydrogenase FadE5 I 29 60.5 5.2 312 20 MAP0783c Rv0853c -1.26 Indolepyruvate decarboxylase Pdc GHR 32 51.9 6.9 151 8 MAP3424c Rv3303c -1.39 Flavoprotein disulfide reductase LpdA C 33 55.4 6.0 230 16 * MAP2004 Rv2251 2.15 Putative
alkyldihydroxyacetonephosphate synthase
C 34 50.2 4.8 104 8 MAP3673c Rv0234c -1.39 Succinic semialdehyde
dehydrogenase GabD1 C
Appendix
change Putative function RCN COG
40 53.0 5.2 115 9 MAP1557c Rv1844c -1.23 6-phosphogluconate
dehydrogenase Gnd G
41 53.0 5.2 117 10 MAP2671c Rv1121 1.81 Glucose-6-phosphate
1-dehydrogenase Zwf G
42 53.7 5.3 97 7 MAP3413 Rv3293 -1.37 Aldehyde dehydrogenase AldB C 43 60.6 5.3 148 9 MAP0891c Rv0946c -1.39 Glucose-6-phosphate isomerase Pgi G 44 55.1 5.3 211 15 MAP1701c Rv1722 -1.15 Biotin carboxylase-like protein I 46 58.1 5.5 112 7 MAP3146c Rv3068c -1.24 Phosphoglucomutase PgmA G 47 46.0 5.8 148 10 MAP3956 Rv0462 -1.39 Dihydrolipoamide dehydrogenase C 50 48.7 5.1 188 13 MAP3204 Rv3148 1.71 NADH dehydrogenase subunit D NuoD C 51 43.7 5.2 89 7 MAP4143 Rv0685 3.34 Elongation factor Tu Tuf J 52 49.8 5.4 151 11 MAP2693 Rv1098c 1.33 Fumarate hydratase Fum C 53 47.1 5.5 103 7 MAP4256 -1.01 Putative carbohydrate kinase G 54 45.0 5.8 160 8 MAP2699c Rv1093 1.11 Serine hydroxymethyltransferase GlyA E 55 48.2 5.5 82 6 MAP0697 / Putative luciferase-like
monooxygenase C
56 46.6 5.6 126 10 MAP3455c Rv3339c -1.01 Isocitrate dehydrogenase 1 Icd1 C 57 44.3 6.2 91 6 MAP3651c Rv0215c -1.22 Putative acyl-CoA
dehydrogenase FadE3_2 I
57 47.4 5.8 115 7 MAP3692c Rv0242c -1.22 3-ketoacyl-(acyl-carrier-protein)
reductase FabG4 IQR
58 47.0 6.2 149 11 MAP3693 Rv0243 1.75 Acetyl-CoA acetyltransferase FadA2 I
59 38.4 6.3 81 6 Fibrinogen [Bos taurus].
60 40.4 4.7 172 12 MAP1177c Rv1448c 1.73 Transaldolase Tal G 61 42.5 4.8 196 13 MAP1165 1.42 Phosphoglycerate kinase Pgk G 62 43.2 5.0 141 11 MAP0896 Rv0951 1.58 Succinyl-CoA synthetase subunit
beta SucC C
63 38.4 4.9 152 10 MAP2008 Rv2259 1.28 S-(hydroxymethyl)mycothiol
dehydrogenase AdhE2 C 64 33.4 5.0 87 4 MAP1563c Rv1855c 1.3 Uncharacterised protein C 65 40.2 5.0 144 10 MAP2436c Rv1323 2.05 Acetyl-CoA acetyltransferase FadA4 I 66 42.7 5.0 110 9 MAP0789 Rv0859 -1.23 Acetyl-CoA acetyltransferase FadA_1 I 67 42.0 5.2 66 † 9 MAP3392c Rv3274c 4.32 Acyl-CoA dehydrogenase FadE25_4 I 67 42.8 5.2 75 8 MAP2227 2.17 Putative acyl-CoA
dehydrogenase FadE17 I 68 41.2 5.2 119 10 MAP2938c Rv2868c 1.4 4-hydroxy-3-methylbut-2-en-1-yl
diphosphate synthase (IspG) GcpE I 69 42.0 5.2 105 9 MAP3392c Rv3274c 4.32 Acyl-CoA dehydrogenase FadE25_4 I 70 43.9 5.2 244 18 MAP1367 Rv1658 1.12 Argininosuccinate synthase ArgG E
71 40.5 5.2 100 8 * MAP3478 2.42 Trehalose-phosphatase OtsB2 G 72 43.7 5.2 143 18 MAP4143 Rv0685 1.27 Elongation factor Tu Tuf J
73 28.0 4.6 140 10 MAP1889c Rv2145c 2.305 Putative cell division protein Wag31 D 74 34.7 4.7 95 6 MAP1942c Rv2202c 1.56 Putative carbohydrate kinase /
adenosine kinase CbhK G 76 23.7 4.8 115 7 MAP1688 3.62 Citrate lyase subunit beta /
citryl-CoA lyase G
77 39.4 5.0 153 11 MAP2228 1.49 Putative acyl-CoA
dehydrogenase I
Supplementary Table S 9-7 cont.
change Putative function RCN COG
78 55.8 4.9 79 7 MAP4265 Rv3417c 2.63 Chaperonin GroEL1 GroEL1 O 78 36.1 5.0 81 7 MAP3668c Rv0230c 2.63 Putative
phosphotriesterase-related protein R
79 40.2 5.1 78 5 MAP0595c Rv0761c 1.6
S-(hydroxymethyl)glutathione dehydrogenase / alcohol
dehydrogenase AdhB C
82 36.2 5.2 89 6 MAP1164 Rv1436 -2.1 Glyceraldehyde-3-phosphate
dehydrogenase Gap G
83 36.7 5.3 183 11 * MAP2670c Rv1122 2.48 6-phosphogluconate
dehydrogenase-like protein Gnd2 G 85 42.3 5.6 154 9 MAP0827c Rv0889c 1.68 Citrate synthase CitA C 86 43.9 5.4 78 7 MAP0445c Rv3618 1.2 Putative luciferase-like
monooxygenase C
88 44.3 6.2 116 13 MAP3651c Rv0215c -1.43 Putative acyl-CoA
dehydrogenase FadE3_2 I
89 44.3 6.2 108 7 MAP3651c Rv0215c -1.68 Putative acyl-CoA
dehydrogenase FadE3_2 I
90 44.3 6.2 76 4 MAP3651c Rv0215c -3.7 Putative acyl-CoA
dehydrogenase FadE3_2 I
91 40.3 6.1 151 10 MAP0841 Rv0906 / Beta-lactamase superfamily
domain R
92 36.8 5.3 172 13 MAP4210 1.87 Putative FAE1/Type III
polyketide synthase-like protein I 93 35.1 5.4 90 7 MAP1613c / Alcohol dehydrogenase AdhA_2 R 94 37.3 5.7 160 12 MAP0812 1.19 Putative glutathione S-transferase O 95 41.7 5.7 148 10 MAP0244 Rv3778c 1.13 Putative Selenocysteine
lyase/Cysteine desulfurase E 96 35.4 5.9 176 13 MAP1361 Rv1652 1.29
N-acetyl-gamma-glutamyl-phosphate reductase ArgC E 97 39.3 5.9 223 18 * MAP0658c Rv0824c / Acyl-[acyl-carrier-protein]
desaturase DesA1 (-)
98 33.8 6.2 97 7 MAP4134 Rv0673 4.1 Enoyl-CoA hydratase EchA4 I 99 38.1 6.1 105 8 MAP2909c Rv2841c Transcription elongation factor
(NusA) NusA K
100 33.4 5.3 78 5 MAP3190 Rv3141 1.28 Putative NADPH2:quinone
reductase FadB4 CR
102 27.8 4.7 73 7 MAP3061c Rv3029c 1.72 Putative electron transfer
flavoprotein beta subunit FixA C 103 43.7 5.2 109 10 MAP4143 Rv0685 1.14 Elongation factor Tu Tuf J
1.67 Enoyl-CoA hydratase EchA8_1 I 116 30.3 6.3 134 8 MAP4227c Rv3463 1.51 Putative luciferase-like
monooxygenase C
Appendix
170
Supplementary Table S 9-7 cont.
Spot
# MW
[Da] pI Score
‡
Matched
peptides Locus tag H37Rv Fold
change Putative function RCN COG
117 26.5 6.2 96 9 MAP2637c Rv1144 1.15 Putative NAD dependent
epimerase/dehydratase family IQR 118 24.3 5.1 93 7 MAP4102c Rv0632c -1.27
Putative enoyl-CoA hydratase/isomerase
(MAPs_39340) EchA3 I
119 23.0 5.6 76 4 MAP0187c Rv3846 1.77 Putative superoxide dismutase SodA P 120 20.6 5.4 98 5 MAP2945c Rv2882c / Ribosome recycling factor Frr J 122 17.6 5.1 78 4 MAP0068 -1.27 Single-strand DNA-binding
protein Ssb L
For further information see legend for Supplementary Table S 9-1. † Protein has given a significant hit excised at similar positions from other 2D gels. * Peptides have been matched by MALDI-TOF/TOF.
9.3. Figures
Fig. 1-1. Holstein–cows with clinical signs of Johne’s disease and mucosal scraping
from M. avium subsp. paratuberculosis containing intestinal peaces... 25
Fig. 1-2. Cultivation of M. avium subsp. paratuberculosis... 26
Fig. 1-3. Schematic workflow for the isolation and enrichment of M. avium subsp. paratuberculosis from gut samples. ... 28
Fig. 1-4. Pictures of M. avium subsp. paratuberculosis enrichment from intestinal mucosa... 29
Fig. 1-5. SDS-PAGE and GeLC-MS/MS of mucosa-and culture-derived cytoplasm. ... 33
Fig. 4-1. Functional classification of proteins identified according to the database Cluster of Orthologous Groups of Proteins (COGs). ... 54
Fig. 4-2. Venn diagrams for the distribution of M. avium subsp. paratuberculosis proteins identified in mucosa- and culture-derived cytoplasm, as detected by LC-orbitrap-MS/MS. ... 55
Fig. 4-3. COG clustering of M. avium subsp. paratuberculosis proteins identified in mucosa- and culture-derived cytoplasm from cow 2 and cow 3, respectively... 56
Fig. 4-4. Proteins of bovine origin in 2D DIGE and 2D GE... 57
Fig. 4-5. Comparative and preparative 2D gelelectrophoresis of in vivo and in vitro grown M. avium subsp. paratuberculosis with protein identification of similar expressed proteins between MDC and CDC. ... 58
Fig. 4-6. Proteins associated with host-induced stress conditions. ... 63
Fig. 4-7. Proteins associated with lipid transport and metabolism... 64
Fig. 4-8. Proteins associated with carbohydrate transport and metabolism. ... 65
Fig. 4-9. Proteins associated with energy production and conversion. ... 66
Fig. 4-10. Schematic of the metabolic pathway network of M. avium subsp. paratuberculosis... 67
9.4. Supplementary figures Supplementary Fig. S 4-1. Representative 2D gels of cytoplasmic protein fractions of mucosa-derived M. avium subsp. paratuberculosis from cow 2 and cow 3. ... 87
Supplementary Fig. S 9-1. Protein spots from 2D GE with MDC (1a) and 2D DIGE with MDC vs. CDC (1b) from cow 2 (batch 1)... 147
Appendix
172
Supplementary Fig. S 9-2. Protein spots from 2D GE with MDC (2a) and 2D DIGE
with MDC vs. CDC (2b) from cow 3 (batch 1)... 149 Supplementary Fig. S 9-3. Protein spots from 2D GE with MDC (3a) and 2D DIGE
with MDC vs. CDC (3b) from cow 3 (technical repeat batch 1). ... 151 Supplementary Fig. S 9-4. Protein spots from 2D GE with MDC (4a) and 2D DIGE
with MDC vs. CDC (4b) from cow 2 (batch 2)... 154 Supplementary Fig. S 9-5. Protein spots from 2D GE with MDC (5a) and 2D DIGE
with MDC vs. CDC (5b) from cow 3 (batch 2)... 157 Supplementary Fig. S 9-6. Protein spots from 2D GE with MDC (6a) and 2D DIGE
with MDC vs. CDC (6b) from cow 2 (batch 3)... 160 Supplementary Fig. S 9-7. Protein spots from 2D GE with MDC (7a) and 2D DIGE
with MDC vs. CDC (7b) from cow 3 (batch 3)... 165
9.5. Tables
Table 4-1. Shared principles of major metabolic pathways of in vivo and in vitro grown M. avium subsp. paratuberculosis as identified by 2D DIGE/2D GE and
subsequent MALDI-TOF analysis in MDC and CDC. ... 61
9.6. Supplementary tables
Supplementary Table S 4-1. Identified proteins excised from corresponding regions at
Coomassie stained preparative 2D gel with MDC from cow2 and cow 3. ... 85 Supplementary Table S 4-2. Proteins quantified by 2D DIGE and identified by 2D GE
and subsequent MALDI-TOF analysis... 88 Supplementary Table S 4-3. Proteins identified in MDC (97), as detected by GeLC-MS. ... 91 Supplementary Table S 4-4. Proteins identified in CDC (129), as detected by
GeLC-MS. ... 93 Supplementary Table S 4-5. Proteins identified in each MDC and CDC (521),
respectively, as detected by LC-orbitrap-MS/MS... 96 Supplementary Table S 4-6. Proteins solely detected in MDC of both strains (41), as
detected by LC-orbitrap-MS/MS... 99 Supplementary Table S 9-1. Identified proteins excised from corresponding regions at
Coomassie stained preparative 2D gel with MDC from cow2... 148
Supplementary Table S 9-2. Identified proteins excised from corresponding regions at
Coomassie stained preparative 2D gel with MDC from cow3... 150 Supplementary Table S 9-3. Identified proteins excised from corresponding regions at
Coomassie stained preparative 2D gel with MDC from cow3 (technical repeat of 2D GE/2D DIGE from Fig. 2a, b and Table 2). ... 153 Supplementary Table S 9-4. Identified proteins excised from corresponding regions at
Coomassie stained preparative 2D gel with MDC from cow2... 156 Supplementary Table S 9-5. Identified proteins excised from corresponding regions at
Coomassie stained preparative 2D gel with MDC from cow3... 159 Supplementary Table S 9-6. Identified proteins excised from corresponding regions at
Coomassie stained preparative 2D gel with MDC from cow2... 162 Supplementary Table S 9-7. Identified proteins excised from corresponding regions at
Coomassie stained preparative 2D gel with MDC from cow2... 162 Supplementary Table S 9-7. Identified proteins excised from corresponding regions at