immune profiling with a salmonella typhi antigen ... · pdf fileimmune profiling with a...
TRANSCRIPT
Immune profiling with a Salmonella Typhi antigen microarray identifies new diagnostic
biomarkers of human typhoid
Li Liang1, Silvia Juarez1, Tran Vu Thieu Nga2, Sarah Dunstan2, Rie Nakajima-Sasaki1, D.
Huw Davies1, Stephen McSorley3, Stephen Baker2, Philip L. Felgner1
1 Department of Medicine, Division of Infectious Diseases, University of California, Irvine,
CA 92697
2Centre for Tropical Medicine, Oxford University Clinical Research Unit, 190 Ben Ham Tu,
Quan 5, Ho Chi Minh City, Vietnam
3Center for Comparative Medicine, Department of Anatomy, Physiology and Cell Biology,
School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Supplementary figures and tables
Supplementary Figure S1. Construction of a S.enterica serovar Typhi Protein Microarray. Arrays were
printed containing >2700 S.Typhi proteins, positive and negative control spots. Each array contains
positive control spots printed from 4 serial dilutions of human and mouse IgG, 2 serial dilutions of human
IgM, 2 serial dilutions of EBNA1 protein, and "No DNA" negative control spots. The array was probed
with anti-HA or anti-His antibody, 96% of the protein spots were positive for the HA or His tag. The
arrays were read in a laser confocal scanner. The signal intensity of each antigen is represented by
rainbow palette of blue, green, red and white by increasing signal intensity. White spots are with saturated
maximum signal intensity (~65000). Red spots are with near maximum signal intensity.
Supplementary Figure S2. The mean IgG reactivity of the antigens was compared between the acute
typhoid patients in Vietnam and Non-typhoidal Salmonellosis patients in Africa. Antigens with Benjamini
Hochberg corrected p-value less than 0.05 are organized to the left and cross-reactive antigens to the right.
Supplmentary Table S1. Serodiagnostic IgG antigens for human typhoid patients in Vietnam
ID Symbol COGs Product Description homologs
identified
Serodiagnostic,
NTS vs
Control
Serodiagnostic,
Typhoid vs
NTS
t1477 hlyE - hemolysin E Y Y
t1111 cdtB - putative toxin-like protein Y Y
t4052 fkpA COG0545O FKBP-type peptidyl-prolyl cis-
trans isomerase
t1128 - COG0071O putative heat shock protein Y
t3710 hslS COG0071O heat shock chaperone IbpB
t1459 - COG3049M putative secreted hydrolase Y Y
t3515 - COG0737F hypothetical protein t3515
t1285 ssaP - putative type III secretion
protein Y
t1224 nlpC COG0791M putative lipoprotein
t3065 hypA COG0375R hydrogenase nickel
incorporation protein HybF
t2970 - - hypothetical protein t2970
t0536 nuoA COG0838C NADH dehydrogenase subunit
A
t1814 hpcG COG3971Q 2-oxo-hepta-3-ene-1,7-dioic
acid hydratase Y
t4322 - COG5484S terminase subunit
t4253 pilK - hypothetical protein t4253
t3904 yhjJ COG0612R putative zinc-protease precursor
Supplementary Table S2. Cross-reactive IgG antigens for human typhoid patients in Vietnam
ID Symbol COGs Product Description
Serodiagnostic
in NTS
t2763 sitA COG0803P iron transport protein, periplasmic-binding protein
t4225 phoN COG0671I nonspecific acid phosphatase precursor Y
t2787 sipB - pathogenicity island 1 effector protein
t1557 - - hypothetical protein t1557
t0500 dedD COG3147S hypothetical protein t0500
t2129 tolA COG3064M cell envelope integrity inner membrane protein TolA
t0623 sspH COG4886S secreted effector protein
t2561 safA - lipoprotein
t0247 rcsF - outer membrane lipoprotein
t2229 rlpA COG0797M rare lipoprotein A
t3828 sapB COG5295UW putative autotransporter
t1496 - COG3391S ATP-binding protein
t2941 - - hypothetical protein t2941 Y
t1649 tonB COG0810M transport protein TonB
t3116 - COG2960S hypothetical protein t3116
t3709 hslT COG0071O heat shock protein IbpA
t0918 fliC COG1344N flagellin
t2126 ybgF COG1729S tol-pal system protein YbgF Y
t3119 - COG2268S hypothetical protein t3119 Y
t1714 - COG5633R putative lipoprotein
t2128 tolB COG0823U translocation protein TolB
t1856 pqiB COG3008R paraquat-inducible protein B
t1495 - - putative lipoprotein
t1529 - COG1304C putative glycolate oxidase
t2605 mltD COG0741M membrane-bound lytic murein transglycosylase D
t2274 fepB COG4592P iron-enterobactin transporter periplasmic binding protein
t0180 yadE COG0726G hypothetical protein t0180
t2799 invE - cell invasion protein
t4166 - - large repetitive protein
t1376 - - hypothetical protein t1376
t3755 mgtB COG0474P magnesium transport ATPase, P-type 2
t0210 htrA COG0265O serine endoprotease
t0995 yebG COG3141S DNA damage-inducible protein YebG
t4456
cpdB COG0737F
bifunctional 2',3'-cyclic nucleotide 2'-
phosphodiesterase/3'-nucleotidase periplasmic precursor
protein
t2636 grpE COG0576O heat shock protein GrpE
t4239 pilL - hypothetical protein t4239
t2785 sipD - pathogenicity island 1 effector protein
t3426 - - putative regulatory protein
t2648 - - hypothetical protein t2648
t1992 ybjY COG0845M macrolide transporter subunit MacA
t4268 - - hypothetical protein t4268
t2786 sipC - pathogenicity island 1 effector protein
t1743 flgE COG1749N flagellar hook protein FlgE
t3234 - COG3117S hypothetical protein t3234
t2826 nlpD COG0739M lipoprotein NlpD
t2415 yajG COG3056M hypothetical protein t2415
t4399 yjeP COG3264M hypothetical protein t4399
t3581 - - putative lipoprotein
t0248 metQ COG1464P DL-methionine transporter substrate-binding subunit
t1187 - COG3678UNTP periplasmic protein
t1449 - COG4319S hypothetical protein t1449 Y
t1658 oppA COG4166E periplasmic oligopeptide-binding protein precursor
t1594 pspB - phage shock protein B
t2449 yajI - hypothetical protein t2449
t1583 mppA COG4166E periplasmic murein peptide-binding protein MppA
t4544 - COG1205R DEAD-box helicase-related protein
t1012 yjcS COG2015Q putative hydrolase
t2017 potH COG1176E
putrescine transporter subunit: membrane component of
ABC superfamily
t3362 hemY COG3071H putative protoheme IX biogenesis protein
t3199 - COG4785R lipoprotein NlpI
t0095 surA COG0760O peptidyl-prolyl cis-trans isomerase SurA
t2734 srlE COG3732G glucitol/sorbitol-specific IIBC component of PTS system
t0112 tbpA COG4143H thiamine transporter substrate binding subunit
t3148 - - hypothetical protein t3148
t2394 - COG3126S lipoprotein
t0340 sinI - hypothetical protein t0340
t4274 - - hypothetical protein t4274
t0903 fliK COG3144N flagellar hook-length control protein
t2583 - COG3521S lipoprotein
t1850 ompA COG2885M outer membrane protein A Y
t0906 fliH COG1317NU flagellar assembly protein H
t3585 fdoI COG2864C formate dehydrogenase-O subunit gamma
t1328 - - hypothetical protein t1328
t0549 - COG0835NT putative receptor/regulator protein
t0754 wza COG1596M putative polysaccharide export protein
t3698 yhjA COG1858P cytochrome c peroxidase
t1147 aroQ COG1605E chorismate mutase
t2591 - COG0542O ClpB-like protein
t0820 pduT COG4577QC putative propanediol utilization protein PduT
t0013 dnaJ COG0484O chaperone protein DnaJ
t4547 - COG3440V hypothetical protein t4547
t1377 - - putative lipoprotein
t3078 exbD COG0848U biopolymer transport protein ExbD
t0037 - COG3119P putative secreted sulfatase
t3280 yhcQ COG1566V p-hydroxybenzoic acid efflux subunit AaeA
t0908 fliF COG1766NU flagellar MS-ring protein
t0429 - COG3115D cell division protein ZipA
t3187 - COG2823R hypothetical protein t3187
t0629 - - hypothetical protein t0629
t2220 rlpB COG2980M LPS-assembly lipoprotein RlpB
t1349 - - putative bacteriophage tail fiber assembly protein
t0707 stcD - hypothetical protein t0707
t3503 argC COG0002E N-acetyl-gamma-glutamyl-phosphate reductase
t1503 srfA - putative virulence effector protein
t3948 - - hypothetical protein t3948
t0029 - COG1651O hypothetical protein t0029
t2516 - - hypothetical protein t2516
t3705 dsbE COG0526OC thiol:disulfide interchange protein
t4616 smp COG3726R hypothetical protein t4616
t3041 - - hypothetical protein t3041
t3011 pilT COG2805NU Type II secretion, ATP-binding, protein
t3970 ggt COG0405E gamma-glutamyltranspeptidase
t0955 motB COG1360N flagellar motor protein MotB
t3668 pstA COG0581P phosphate transporter permease subunit PtsA
t1468 - - hypothetical protein t1468
t0294 yfhG - hypothetical protein t0294
t2491 - COG3468MU puative autotransporter/virulence factor
t1123 - - lysozyme inhibitor
t0822 pduQ COG1454C putative propanol dehydrogenase
t4415 hflC COG0330O FtsH protease regulator HflC
t0705 stcB COG3121NU putative fimbrial chaperone protein
Supplementary Table S3. Serodiagnostic IgM antigens for human typhoid patients in Vietnam.
ID Symbol COGs Product Description
t3116 - COG2960S hypothetical protein t3116
t1594 pspB - phage shock protein B
t1376 - - hypothetical protein t1376
t2538 yafK COG3034S hypothetical protein t2538
t4239 pilL - hypothetical protein t4239
t0717 - - hypothetical protein t0717
t2449 yajI - hypothetical protein t2449
t2126 ybgF COG1729S tol-pal system protein YbgF
t0058 oadG COG3630C oxaloacetate decarboxylase subunit gamma
t2864 - COG3609K hypothetical protein t2864
t1855 - COG3009S putative lipoprotein
t1153 - COG2261S hypothetical protein t1153
t1548 - - putative lipoprotein
t3413 - - lipoprotein
t3709 hslT COG0071O heat shock protein IbpA
t2134 - COG4890S hypothetical protein t2134
t1039 - - hypothetical protein t1039
t2941 - - hypothetical protein t2941
t4290 - - hypothetical protein t4290
t2415 yajG COG3056M hypothetical protein t2415
t3324 tatA COG1826U twin arginine translocase protein A
t4440 yjfY - hypothetical protein t4440
t1768 - COG5645R hypothetical protein t1768
t1128 - COG0071O putative heat shock protein
t4268 - - hypothetical protein t4268
t4293 - - hypothetical protein t4293
t0458 - - hypothetical protein t0458
t2633 corE COG4137R hypothetical protein t2633
t0294 yfhG - hypothetical protein t0294
t3497 yijD - hypothetical protein t3497
t4430 yjfO - hypothetical protein t4430
t3561 cpxA COG0642T two-component sensor protein
t4312 - - putative regulatory protein
t3703 ccmE COG2332O cytochrome c-type biogenesis protein CcmE
t3828 sapB COG5295UW putative autotransporter
t2220 rlpB COG2980M LPS-assembly lipoprotein RlpB
t3970 ggt COG0405E gamma-glutamyltranspeptidase
t2332 fdrA COG0074C membrane protein FdrA
t2588 - COG3516S hypothetical protein t2588
t3948 - - hypothetical protein t3948
t3862 - - putative lipoprotein
t2636 grpE COG0576O heat shock protein GrpE
t3426 - - putative regulatory protein
t1474 - - putative secreted protein
t2832 ftsB COG2919D cell division protein FtsB
t2903 ptr COG1025O protease III precursor
t3605 ompL - outer membrane porin L
t0075 caiT COG1292M L-carnitine/gamma-butyrobetaine antiporter
t0614 ccmE COG2332O cytochrome c-type biogenesis protein CcmE
t0029 - COG1651O hypothetical protein t0029
t3503 argC COG0002E N-acetyl-gamma-glutamyl-phosphate reductase
t1898 - - putative secreted protein
t4605 - - hypothetical protein t4605
t1057 - COG1214O hypothetical protein t1057
t2023 ybjC - hypothetical protein t2023
t2813 - COG0583K LysR family transcriptional regulator
t1779 csgE - curli assembly protein CsgE
t3097 - COG3111S hypothetical protein t3097
t3055 exuT COG2271G hexuronate transporter
t1349 - - putative bacteriophage tail fiber assembly protein
t2235 tatE COG1826U twin arginine translocase protein E
t0097 djlA COG1076O Dna-J like membrane chaperone protein
t2107 ybhT - hypothetical protein t2107
t3668 pstA COG0581P phosphate transporter permease subunit PtsA
t0488 - - putative lipoprotein
t1591 pspE COG0607P thiosulfate:cyanide sulfurtransferase
t4564 - COG3314S hypothetical protein t4564
t2516 - - hypothetical protein t2516
t2210 gltK COG0765E glutamate/aspartate transport system permease protein GltK
t1566 - - hypothetical protein t1566
t1598 sapB COG4168V peptide transport system permease protein SapB
t2655 - - hypothetical protein t2655
t4555 - COG0412Q hypothetical protein t4555
t0472 vacJ COG2853M VacJ lipoprotein precursor
t4138 malM - maltose regulon periplasmic protein
t1377 - - putative lipoprotein
t1313 slyB COG3133M outer membrane lipoprotein SlyB precursor
Supplementary Table S4. Cross-reactive IgM antigens for human typhoid patients in Vietnam.
ID Symbol Product
t4239 pilL hypothetical protein t4239
t1890 - putative bacteriophage protein
t2975 visB 2-octaprenyl-6-methoxyphenyl hydroxylase
t0058 oadG oxaloacetate decarboxylase subunit gamma
t3108 - hypothetical protein t3108
t1153 - hypothetical protein t1153
t1855 - putative lipoprotein
t3709 hslT heat shock protein IbpA
t1039 - hypothetical protein t1039
Supplementary Table S5. Enrichment analysis on Clusters of Orthologous Groups (COGs) of IgG antigens
identified in this study. Classifications over-represented (enriched) among ‘hits’ have foldenrich values >1 and
those under-represented have values <1. The significance of enrichment values were also calculated using Fisher’s
exact test in the R environment. A p-value of <0.05 indicated a significant fold-enrichment. Significant over-
representation and under-representation are underlined.
proteins Serodominant Serodiagnostic
COG Definition on chip Hits FoldEnrich p-value Hits FoldEnrich p-value
C Energy production and conversion 126 1 0.3 3.69E-01 0 0.0 1.00E+00
D Cell division and chromosome partitioning 13 0 0.0 1.00E+00 0 0.0 1.00E+00
E Amino acid transport and metabolism 168 1 0.3 1.87E-01 0 0.0 1.00E+00
F Nucleotide transport and metabolism 16 2 5.2 5.53E-02 0 0.0 1.00E+00
G Carbohydrate transport and metabolism 187 1 0.2 8.69E-02 0 0.0 1.00E+00
H coenzyme metabolism 46 0 0.0 6.28E-01 0 0.0 1.00E+00
I Lipid metabolism 36 1 1.2 5.87E-01 0 0.0 1.00E+00
J Translation, ribosomal structure and
biogenesis 45 0 0.0 6.27E-01 0 0.0 1.00E+00
K Transcription 153 0 0.0 5.07E-02 0 0.0 1.00E+00
L DNA replication, recombinationand repair 72 0 0.0 4.19E-01 0 0.0 1.00E+00
M Cell envelope biogenesis, outer memberane 167 12 3.0 5.11E-04 1 2.2 3.69E-01
N Cell motility and secretion 109 3 1.1 7.46E-01 0 0.0 1.00E+00
O Posttranslational modification, protein
turnover, chaperones 101 7 2.9 9.97E-03 3 11.1 1.86E-03
P Inorganic ion transport and metabolism 143 4 1.2 7.76E-01 0 0.0 1.00E+00
Q Secondary metabolites biosynthesis, transport
and catabolism 39 1 1.1 6.16E-01 0 0.0 1.00E+00
R General function prediction only 236 5 0.9 1.00E+00 0 0.0 1.00E+00
S Function unknown 228 9 1.6 1.16E-01 2 3.3 1.20E-01
T Signal transduction mechanisms 126 0 0.0 7.36E-02 0 0.0 1.00E+00
U Intracellular trafficking and secretion 122 4 1.4 5.37E-01 0 0.0 1.00E+00
V Defense mechanisms 37 0 0.0 1.00E+00 0 0.0 1.00E+00
W Extracellular Structure 1 1 41.5 2.41E-02 0 0.0 1.00E+00
Other COGs 1 0 0.0 1.00E+00 0 0.0 1.00E+00
Not in COGs 814 20 1.0 8.94E-01 2 0.9 1.00E+00
Total 2986 72 8
Supplementary Table S6. Enrichment analysis on computationally predicted features of IgG
antigens identified in this study.
proteins Serodominant Serodiagnostic
Computational Predictions on chip Counts FoldEnrich p-value Counts FoldEnrich p-value
TMHMM=0 1820 51 1.1 2.44E-01 6 1.1 1.00E+00
TMHMM=1 270 14 2.1 7.12E-03 2 2.5 1.84E-01
TMHMM=2-5 290 2 0.3 2.90E-02 0 0.0 1.00E+00
TMHMM=6-10 211 1 0.2 4.02E-02 0 0.0 1.00E+00
TMHMM>10 133 1 0.3 2.58E-01 0 0.0 1.00E+00
Signal P>=0.7 712 38 2.1 2.40E-07 5 2.4 3.27E-02
Signal P<0.7 2012 31 0.6 2.40E-07 3 0.5 3.27E-02
pSortb Cytoplasmic 627 4 0.3 2.16E-04 0 0.0 2.11E-01
pSortb Cytoplasmic Membrane 661 4 0.2 8.51E-05 0 0.0 2.11E-01
pSortb Extracellular 23 3 5.2 1.92E-02 0 0.0 1.00E+00
pSortb Outer Membrane 63 2 1.3 6.73E-01 0 0.0 1.00E+00
pSortb Periplasmic 119 11 3.7 1.48E-04 1 2.9 3.01E-01
pSortb Unknown 1231 45 1.4 8.44E-04 7 1.9 2.66E-02
pI 0-5 282 10 1.4 2.33E-01 1 1.2 5.83E-01
pI 5-9 1610 46 1.1 2.16E-01 6 1.3 4.84E-01
pI 9-14 832 13 0.6 3.39E-02 1 0.4 4.48E-01
Total ORFs 2724 69 8
Supplementary Table S7. Enrichment analysis on evidence of expression by Mass Spectrometry of
IgG antigens identified in this study.
proteins Serodominant Serodiagnostic
Evidence of Expression by Mass Spec on chip Hits FoldEnrich p-value Hits FoldEnrich p-value
Expressed and detected with at least 1 peptide 923 41 1.8 1.37E-05 7 2.6 2.85E-03
Expressed and detected with at least 10 peptides 715 38 2.1 4.42E-07 7 3.3 5.19E-04
Expressed and detected with at least 20 peptides 503 33 2.6 1.66E-08 5 3.4 7.22E-03
Expressed and detected with at least 50 peptides 206 15 2.9 1.31E-04 3 5.0 1.80E-02
Expressed and detected with at least 100 peptides 77 6 3.1 1.22E-02 1 4.4 2.05E-01
Not expressed 1801 28 0.6 1.37E-05 1 0.2 2.85E-03
Total 2724 69 8