Bovine Gene Expression in Acute Salmonella enerica Serovar Typhimurium Infection Analyzed by MPSS and Microarray.
Sara D. Lawhon1, Sangeeta Khare1, Carlos A. Rossetti1, Cristi L. Galindo4, Robin E. Everts3 , Josely F. Figueiredo1, Jairo E. S. Nunes1, Tamara Gull1,
Christen Haudenschild6, George S. Davidson5, Harold R. Garner4, Ken Drake7, Harris A. Lewin3, Andreas J. Bäumler2, and L. Garry Adams1
1
Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4467;
2
Department of Medical Microbiology and Immunology, College of Medicine, University of California at Davis, CA 95616-8645; 3 Department of Animal Sciences, University of Illinois, Urbana, IL,
61801; 4 Eugene McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical School, Dallas, TX 75390-8591; 5 Sandia National Laboratories, Computation, Computers and Mathematics Center, Albuquerque, NM 87123; 6 Solexa, Inc., Hayward, CA 94545; 7 Serologix, Austin, TX 78730
ABSTRACT
AIM OF THE STUDY
Salmonella enterica Serovar Typhimurium (S. Typhimurium) is the
leading cause of death in humans due to foodborne illness in the United
States. Infection in humans and young calves is characterized by
polymorphonuclear cell influx to the intestinal mucosa and diarrhea. The
clinical similarities between human and bovine infection make the calf an
ideal model for human salmonellosis. Previous studies have shown that
fluid secretion caused by S. Typhimurium is caused by effector proteins
secreted by the Type III Secretion System (T3SS), specifically SipA,
SopA, SopB, SopD, and SopE2. Loss of the genes encoding these
effectors reduces not only fluid secretion in bovine ligated ileal loops but
also Interleukin 8 (IL-8) gene expression. The objective of this study was
to create a profile of host gene expression during acute infection with
wild type S. Typhimurium and also an isogenic sipA sopABDE2 mutant.
This was accomplished using microarray analysis and Massively Parallel
Signature Sequencing (MPSS). A microarray time-course study was
performed by collecting and analyzing samples from bovine ligated ileal
loops over seven time points, 15 minutes, 30 minutes, 1 hour, 2, 4, 8, and
12 hours. MPSS analysis measures signatures from individual mRNA
sequences. MPSS was performed on pooled samples from 1 hour, 2
hours, and 4 hours and identified 5,460 unique signatures. Signatures
included those for genes previously identified in host response to
Salmonella such as chemokine (C-C motif and C-X-C motif) ligand, tumor
necrosis factor receptor superfamily (member A1 and 5), prostaglandin E
synthase, lymphotoxin- and fibronectin 1 as well as signatures for
genes with previously unrecognized roles in host response to
Salmonella infection including dipeptidylpeptidase IV (adenosine
deaminase complexing protein-2), angiotensin receptor 1, angiotensin II
receptor-associated protein, thyroid receptor interacting protein, and
adhesion molecule syndecan-4.
MPSS also revealed evidence of
extensive antisense transcript expression. Signatures identified only in
the wild type inoculated loops may serve as potential biomarkers
specific for early infection.
To identify host responses to Salmonella typhimurium wild type at the transcriptome level and to
compare the responses of the host in the absence of key virulence genes, sipAsopABDE2
Expression Level of Individual Genes as Calculated by MPSS
Sip A
SopA
SopB
SopD
0
SopE2
Unknown
Reduces fluid accumulation.
No effect on invasion
Induction of chloride secretion,
Cytoskeletal rearrangements
Inositol phosphate
phosphatase
Complement &
Coagulation
NONSIGNIFICANT
SIGNATURES
14473
UNRELIABL
E
SIGNIFICAN
T
2225
HIT
GENOME
338
NO HIT
TO
GENOME
1887
RELIABLE
NONSIGNIFICA
NT
5987
Toll-like Receptor
Signaling
UNRELIABLE
NONSIGNIFICA
NT
8486
T-cell Receptor
Signaling
MAPK Signaling
HIT
GENOME
2038
NO HIT
TO
GENOME
3949
HIT
GENOME
1941
NO HIT
TO
GENOME
6545
B-cell Receptor
Signaling
Classification of signatures
Virtual
signature
class
1, 2, 3, 22, 23
12, 13, 14, 15,
16
Reduces fluid accumulation.
No effect on invasion
Fluid Accumulation
5.00
Process
NO HIT
TO
GENOME
11906
Reduces fluid accumulation.
No effect on invasion
FLUID ACCUMULATION
Excision
Apoptosis
Jak-STAT Signaling
Reduces fluid accumulation.
No effect on invasion
Our lab has previously shown1 these five effectors (SipA, SopA, SopB, SopD,
and SopE/E2), act in concert to induce inflammatory changes and fluid
secretion in the bovine ligated ileal loop model of S. typhimurium infection.
We employed gene expression profiling by massively parallel signature
sequencing (MPSS) to evaluate global alterations in in vivo gene expression
of the bovine ileal Peyer’s patch caused by infection with wild type S.
typhimurium strain IR715 (WT) and compared these changes to those elicited
by a sipAsopABDE2 mutant (ZA21) and also by LB broth. MPSS captures and
identifies transcript sequences and analyzes the level of expression of
virtually all genes expressed in a sample by counting the number of individual
mRNA molecules derived from a gene to the level of only a few transcripts
present per one million transcripts reported as transcripts per million (TPM).
Perinatal Calf Ileal Loop Model
Time 0
RELIABLE
SIGNIFICAN
T
24596
HIT
GENOME
12690
Fluid secretion, Stimulation of PMN
transmigration
*
4.00
*
3.00
2.00
1.00
0.00
0.25
0.5
LB negative control
Wild type
sipA sopABDE2
1
2
4
8
MT
1
C-C-L8
IL-1Ra
NOS
Angiotensin
R1
mRNA
orientation
Polyadenylation
feature
Leukocyte
Transendothelial
Migration
Total
Signature
Forward
strand
Poly A
signal/tail
5260
Reverse
strand
Poly A
signal/tail
541
ECM Receptor
Interaction
Calcium Signaling
Pathway
Cytokine-cytokine
Receptor Interaction
Signatures present in one experimental group
Group
LB
Total
Signa
-tures
3,225
Not Annotated
Annotated
Total
>50
TPM
<50
TPM
Total
>50
TPM
<50
TPM
2,234
107
2,127
991
43
948
MT
5,774
3,668
267
3,401
2,106
124
1,982
WT
3,307
2,320
239
2,081
987
60
927
Claudin
Legumain
0.1
Wild Type
Mutant
Expression Level of Individual Genes by Real-Time-PCR
540
1501
Cell Adhesion
Modules
Antigen Processing
and Presentation
Condition
Total
Changed
Genes
Unique to
Condition
MT
3
3
WT
2
2
MT
0
0
WT
2
2
MT
1
1
WT
4
4
MT
1
1
WT
2
2
MT
2
1
WT
7
MT
Common
Between
Conditions
0
0
100
10
1
C-C-L8
IL-1Ra
NOS
Angiotensin
R1
Claudin
Legumain
0.1
Mutant
Total Number
Genes in
Pathway
100
69
Total Number of
Genes on
Pathway
Measured
25
21
Modeling Discovered
Mechanistic Genes
5
7
0
99
24
5
0
99
18
4
NM_205808:Bt.16026:Bt63: CLLD4-like
protein
0
57
5
0.010
0.103
0.975
AW482166:Bt.4637:Bt63: cytochrome c
oxidase subunit VIII, heart
73
25
121
0.075
0.275
0.015
NM_174233:Bt.4539:Bt63: angiotensin
receptor 1 [angiotensin II receptor 1]
178
546
82
0.018
0.109
0.001
79
16
1
0.021
0.988
0.999
0
62
0
0.008
0.000
0.008
AF001262:Bt.3885:Bt63: chloride channel,
calcium activated, family member 3
39
59
4
0.410
0.011
0.003
AB098893:Bt.2981:Bt63: similar to protein
kinase C receptor
58
2
15
0.983
0.047
0.998
NM_174242:Bt.1229:Bt63: apolipoprotein A1
12
80
58
0.011
0.029
0.473
NM_175797:Bt.4757:Bt63: Rho GDP
dissociation inhibitor (GDI) beta
0
104
0
0.004
0.000
0.004
NM_173880:Bt.113:Bt63: histone H4
2
86
0
0.974
0.479
0.005
70
71
2
0.968
0.001
0.001
0
83
0
0.005
0.000
0.005
AW266930:Bt.6768:Bt63: secreted protein,
acidic, cysteine-rich [osteonectin]
62
81
0
0.565
0.008
0.006
AB099044:Bt.2878:Bt63: polyubiquitin
90
0
31
0.005
0.073
0.022
NM_181003:Bt.5456:Bt63: aquaporin 4
16
53
0
0.070
0.058
0.011
NM_181003:Bt.5456:Bt63: aquaporin 4
30
44
113
0.465
0.032
0.084
NM_174799:Bt.23174:Bt63: invariant chain
13
19
63
0.522
0.025
0.059
NM_173893:Bt.5269:Bt63: beta-2microglobulin
33
250
2
0.002
0.006
0.000
BE484650:Bt.5269:Bt63: beta-2microglobulin
18
104
14
0.012
0.641
0.007
BF707251:Bt.4751:Bt63: B.taurus mRNA for
MHC class II, clone R1-1
82
0
0
0.006
0.006
0.000
164
49
0
0.034
0.002
0.012
CF614249:Bt.8957:Bt63: chemokine (C-X-C
motif) receptor 4
0
55
0
0.010
0.000
0.010
NM_174501:Bt.2598:Bt63: arachidonate 15lipoxygenase
54
16
0
0.061
0.010
0.061
NM_177432:Bt.10077:Bt63: interferon
responsive factor 1
74
356
56
0.006
0.543
0.003
NM_174346:Bt.504:Bt63: heat shock 10kDa
protein 1 (chaperonin 10)
NM_174077:Bt.12916:Bt63: glutathione
peroxidase 3 (plasma)
D37956:Bt.8552:Bt63: Bovine BoLA-DRA
mRNA for MHC class II BoLA-DR-alpha,
partial cds, clone KR1
P-Val:
LB vs
WT
P-Val:
MT vs
WT
Transcripts present in S.typhimurium
infected group only
(POTENTIAL BIOMARKERS)
LB
MT
WT
Orphan Signature 1
0
0
6530
6
Orphan Signature 2
0
0
3893
2
1
Orphan Signature 3
0
0
1872
WT
3
2
Orphan Signature 4
0
0
1318
MT
1
0
Orphan Signature 5
0
0
1308
WT
6
5
Orphan Signature 6
0
0
1216
MT
0
0
Orphan Signature 7
0
0
1175
WT
1
1
Orphan Signature 8
0
0
1162
MT
0
0
Orphan Signature 9
0
0
1076
Orphan Signature 10
0
0
928
1
1
251
72
46
17
17
2
1
209
33
11
0
159
24
6
0
159
24
11
0
159
24
2
159
24
9
WT
1
1
MT
0
0
WT
1
1
MT
0
0
WT
1
1
MT
0
0
WT
1
1
MT
0
0
WT
1
1
0
MT
0
0
0
0
159
24
8
159
24
2
159
24
1
0
CONCLUSIONS
Signature
Bovine Microarray Analysis
Overview
Total RNA samples were collected from bovine ligated ileal
loops inoculated with Luria-Bertani broth, Wild Type S.
Typhimurium, or an isogenic sipA, sopABDE2 mutant at
seven time points (15 minutes, 30 minutes, 1 hour, 2 hours, 4
hours, 8 hours, and 12 hours) from the same four animals that
were used for the MPSS analysis. Bovine microarrays were
hybridized with cDNA generated from this RNA. Analysis of
the gene expression profile from these samples is currently
ongoing.
*Several unique transcripts were identified in the absence of virulence factors thus predicting
novel functions of the five SPI-1 T3SS translocated proteins.
*Several biomarkers were discovered that were solely present in only one experimental group.
*MPSS-generated data also provided valuable insight of natural antisense genes during the
early phase of infection that had not been identified by other genomics tools.
REFERENCES:
1.
2.
WT
AJ688240:Bt.15397:Bt63: ribosomal protein
L9
Pathway Comparison of Significant Changed Genes
Regulation of Actin
Cytoskeleton
GTP/GDP exchange
0
MT
Pathway
TOTAL
NUMBER OF
SIGNATURES
41294
Reduces fluid accumulation
and inflammatory response.
No effect on invasion
Fluid Accumulation
Bacteriology
Electron Microscopy
Histopathology
RNA Extraction
23
Signatures passed via significant and reliable filter
Actin bundling;
via PKC activation
via PEEC secretion
Cytoskeletal rearrangements
WT
10
Wild Type
Bovine ligated ileal loop
Unknown
139
LB
800
511
Cytoskeletal rearrangements
Induces PEEC secretion
Induces PMN transmigration factor
Fluid secretion, PMN influx
WT
28
SIGNIFICANT
SIGNATURES
26821
Biochemical
Activity
443
576
LB
Invasion of the intestinal epithelium by Salmonella requires a type III secretion
system (T3SS) encoded in Salmonella Pathogenicity Island 1 (SPI-1) to
translocate effector proteins into host epithelial cells. Effector proteins, SipB
and SipC, together form a pore in the host cell membrane through which
additional effector proteins are translocated into the host cell cytoplasm. The
functions of some of these proteins are as follow:
Effect on Cells
128
184
MT
AW463153:Bt.1729:Bt63: ferritin, light
polypeptide
Fold Change as compared to control
(normalized against GAPDH)
1501
Differentially down regulated
Signatures
100
Fold change as compared to control
Differentially up regulated
Signatures
LB
P-Val:
LB Vs
MT
Description
Overview of MPSS methodology2
INTRODUCTION
Effector
Antisense transcripts: regulation of
gene expression due to antisense nature
Zhang, S., Santos, R. L., Tsolis, R. M., Stender, S., Hardt, W. D., Bäumler, A. J. & Adams, L. G. (2002) Infect Immun 70, 3843-55.
Brenner, S., Johnson, M., Bridgham, J., Golda, G., Lloyd, D. H., Johnson, D., Luo, S., McCurdy, S., Foy, M., Ewan, M., Roth, R., George, D., Eletr, S., Albrecht, G., Vermaas, E., Williams, S. R., Moon, K.,
Burcham, T., Pallas, M., DuBridge, R. B., Kirchner, J., Fearon, K., Mao, J. & Corcoran, K. (2000) Nat Biotechnol 18, 630-4.
12
Time Post Inoculation (Hours)
Acknowledgements: This work was supported by a Texas A&M University, Life Sciences Task Force grant.
LB control
30 min
WT
30 min
MT
30 min