immune profiling with a salmonella typhi antigen ... · pdf fileimmune profiling with a...

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


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


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


t1992 ybjY COG0845M macrolide transporter subunit MacA


t2786 sipC - pathogenicity island 1 effector protein

t1743 flgE COG1749N flagellar hook protein FlgE


t2826 nlpD COG0739M lipoprotein NlpD

t2415 yajG COG3056M hypothetical protein t2415

t4399 yjeP COG3264M hypothetical protein t4399


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


t2394 - COG3126S lipoprotein

t0340 sinI - hypothetical protein t0340


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


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


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


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


t0029 - COG1651O hypothetical protein t0029


t3705 dsbE COG0526OC thiol:disulfide interchange protein

t4616 smp COG3726R hypothetical protein t4616


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


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


t1594 pspB - phage shock protein B


t2538 yafK COG3034S hypothetical protein t2538

t4239 pilL - hypothetical protein t4239


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



t3413 - - lipoprotein

t3709 hslT COG0071O heat shock protein IbpA





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




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


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




t2636 grpE COG0576O heat shock protein GrpE


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


t3503 argC COG0002E N-acetyl-gamma-glutamyl-phosphate reductase

t1898 - - putative secreted protein



t2023 ybjC - hypothetical protein t2023

t2813 - COG0583K LysR family transcriptional regulator

t1779 csgE - curli assembly protein CsgE


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


t1591 pspE COG0607P thiosulfate:cyanide sulfurtransferase



t2210 gltK COG0765E glutamate/aspartate transport system permease protein GltK


t1598 sapB COG4168V peptide transport system permease protein SapB


t4555 - COG0412Q hypothetical protein t4555

t0472 vacJ COG2853M VacJ lipoprotein precursor

t4138 malM - maltose regulon periplasmic protein


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


t1855 - putative lipoprotein

t3709 hslT heat shock protein IbpA


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.


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.


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




Not expressed 1801 28 0.6 1.37E-05 1 0.2 2.85E-03

Total 2724 69 8

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