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1
te Pas et al – Additional file 5 – Table with summary and detailed results of pathways analysis
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Table networks results
Summary of the pathway analysis. chicken line idfferences-related expression differences and line-specific or general response to Salmonella infection of
pathways and networks of pathways. Network names are highlighted in yellow. Line A is faster growing, higher susceptible to Salmonella than line B.
Network / Pathway
Line
Salmonella infection effects
Conclusions
Remarks / hypotheses
differences
Line A vs
Line A4,5,6
Line B3,4
Line A vs Line
1, 2,3
Line B
B1, 2
Actin cytoskeleton
Line
infection
X
equal
Line A is induced by
Genetic makeup of line B
differences
effect
infection to equal
sufficient for efficient
line B
reaction to Salmonella
* Adherens junction
B>A
↑
↑
A=B
↑A>B
actin polymerization,
specifically line A
* Tight junction
B>A
↑
X
A=B
line A effect
input for Regulation of actin
cytoskeleton
* Focal adhesion
B>A
↑
X
A=B
line A effect
input for Regulation of actin
cytoskeleton, actin
polymerization, and others
* Axon guidance
B>A
↑
↑
A=B
↑A>B
three subpathways leading to
input for Regulation of actin
cytoskeleton
* Leukocyte transendothelial
B>A
↑
X
A=B
line A effect
input for Regulation of actin
migration
cytoskeleton
* Cell adhesion molecules
B>A
↑
(↓)
A=B
mostly line A effect
Interaction with Adherens
junction, Tight junction, and
Leukocyte transendothelial
migration
* WNT signaling
B>A
↑
X
B>A
line A effect not
input from Adherens junction
sufficient to equal line
and Focal adhesion pathways
B
* B-cell receptor signaling
A>B
↑
↓
A=B
line B less genes react,
input for Regulation of actin
but more induced
cytoskeleton
* T-cell receptor signaling
A=B
↑
(↑)
A=B
mostly line A effect
input for Regulation of actin
cytoskeleton
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* Regulation of actin cytoskeleton
Apoptosis mechanism
B>A
no line
differences
↑
up regulation
(↑)
up regulation
A=B
differences
* Apoptosis
* JAK-STAT signaling
A?B
A?B
↑
↑
↑
(↑)
A>B
A>B
* Natural Killer cell mediated
cytotoxicity
* Transforming Growth Factor (TGF)
beta
A?B
↑
(↑)
A>B
B>A
↑
X
B>A
A?B
↑
(↑)
B>A
line
differences
for fatty
acid
metabolism
A=B
down
regulation
down
regulation
B>A, energy
availability
↓
X
B>A
* Fatty acid metabolism
* Glycerolipid metabolism
B>A
A=B
↓
X
↓
X
B>A
A=B
* Pentose phosphate metabolism
A=B
↑↓
X
A=B
* Purine metabolism
B>A
↑
↑
B>A
* Insulin signaling
Other metabolism pathways
* Amino acid metabolism
B>A
↑↓
↑↓
A=B
no line
?
?
effect
* Mitogen-Activated Protein Kinase
(MAPK) signaling
Energy metabolism
* Glycolysis / Gluconeogenesis
line B little effect
apoptosis and anti
apoptosis /
proliferation and
differentiation
effects differ
Stress fibre formation induced
mostly line A effect
cell growth, differentiation; anti
apoptosis
line B: induced release of
cytokines
proliferation, differentiation
line A effect not
sufficient to equal line
B
mostly line A effect
proliferation, differentiation
input from and to other energy
metabolism pathways
line differences
may be some effect on input
for Glycolysis in line A
may be some effect on input
for Glycolysis in line A
input for Pentose phosphate
metabolism
input for Glycolysis
4
** Val, Leu, Ile degradation
differences
B>A
↑-↓
↑-↓
questionable
A>B / B>A
** Gly, Ser, Thr metabolism
** Beta-Alanine metabolism
* Proteasome
* Caprolactam degradation
B>A
A=B
A=B
B>A
X
X
↑-↓
↓
↓
X
X
X
A=B
A=B
A=B
B>A
* Metabolism of Xenobiotics by P450
A=B
↓
↓
A=B
line
differences
A=B
line
differences?
X
line
differences?
X
no overall
effect
A=B
B>A
A=B
↑
X
X
X
A=B
A=B
A=B
X
X
A=B
line
differences
B>A
functionality?
functionality?
functionality?
X
↓
B>A
B>A
B>A
↓
X
↓
↓
B>A
B>A
Immune response pathways
* Cytokine-cytokine receptor
interaction
* Adipocytokine signaling
* Gap junction
* Complement and coagulation
cascades
Other body functional pathways
* Androgen and Estrogen metabolism
* Circadian rhythm
* Bile acid biosynthesis
overall effect uncertain
Pathway shows internal
confusing regulation
porphyrine biosynthesis
many links with other pathways
difference between the
lines increases due to
infection
Salmonella causes
sharp down regulation
no regulation observed
only input is regulated
line differences related?
input from cytokine-cytokine
receptor pathway
no regulation observed
line B effect not
sufficient to equal line
A
Estrogen metabolism
line B effect not
sufficient to equal line
A
intestine functionality
1: A>B: Expression level of the genes in the pathway differ; the pathway is higher expressed in line A than in line B
2: A=B: Expression levels of some genes in the pathway may be different, but this is not sufficient to call a line-specific difference in the expression of the
pathway
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3: A?B: Regulation observed, but direction (up or down) uncertain and variable
4: ↑: up regulation after Salmonella infection; ↓: down regulation after infection
5: (↑): some up regulation observed after Salmonella infection, but less than in the other line; (↓): some down regulation observed after Salmonella infection,
but less than in the other line
6: X: no regulation observed
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Detailed description of Individual pathways to support the data in the Table
Network: Mechanisms of Cytoskeletal changes
Adherens junction: Chicken line differences resulted in a higher expression in line B compared to line A
of the actin polymerization subpathway. Following Salmonella infection both lines show up regulation
of several genes in this pathway, where especially in line A up “Regulation of the actin polymerization”
subpathway was found. This subpathway shows equal expression between the lines after 24 h infection
with Salmonella. It can be concluded that this equal expression level is the result of a higher up
regulation in line A than in line B.
Tight junction: Chicken line differences resulted in a higher expression in line B compared to line A of
the subpathway leading to input for the Regulation of actin cytoskeleton pathway. Following
Salmonella infection line A shows up regulation of the genes in the subpathway leading to input for
the “Regulation of actin cytoskeleton” pathway. No such effect was observed in line B. This subpathway
shows equal expression between the lines after 24 h infection with Salmonella. It can be concluded
that this equal expression level is the result of the up regulation in line A.
There is an input subpathway from the “Adherens junction” pathway. Some up regulation of
gene expression in line A after infection is seen suggesting that the up regulation of the “Adherens
junction” pathway in line A after infection also affects gene expression in the “Tight junction pathway”.
Focal adhesion: Chicken line differences resulted in a higher expression in line B compared to line A for
the subpathways actin polymerization, cell proliferation, cell motility, phosphatidylinositol signalling,
and input for the pathway “Regulation of actin cytoskeleton”. Following Salmonella infection the actin
cytoskeleton related traits are up regulated in line A. No such effect was observed in line B. This
subpathway shows equal expression between the lines after 24 h infection with Salmonella. It can be
concluded that this equal expression levels is the result of the up regulation in line A although some
genes are regulated after infection in line B.
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Axon guidance: The pathway shows three subpathways leading to input for the “Regulation of actin
cytoskeleton” pathway. Chicken line differences resulted in a higher expression of all three
subpathways in line B compared to line A. Following Salmonella infection both lines show up
regulation of the three mentioned subpathways, but this induction is more in line A (more genes
induced) than in line B. These subpathways show equal expression between the lines after 24 h
infection with Salmonella. It can be concluded that this equal expression levels is the result of a higher
up regulation in line A than in line B.
Leukocyte transendothelial migration: Activation of leukocytes leading to transendothelial migration
may be associated with changed cytoskeletal appearance. The pathway consists of gene networks in
both the leukocytes and the endothelial cells and these gene networks are interacting. Chicken line
differences resulted in a higher expression in line B compared to line A for subpathways towards
transendothelial migration and input for the Regulation of actin cytoskeleton pathway. Following
Salmonella infection line A shows up regulation of several genes in both subpathways mentioned
above. This subpathway shows equal expression between the lines after 24 h infection with Salmonella.
It can be concluded that this equal expression levels is the result of the up regulation in line A.
“Cell adhesion molecules” are involved in several steps of the migration. Although many genes
show differential expression after Salmonella infection there is no overall effect suggested.
Cell adhesion molecules: The KEGG database collects a number of different adhesion mechanisms
between cells in this pathway. Interactions with “Adherens junction”, “Tight junction”, and “Leukocyte
transendothelial migration” pathways are suggested at several places. Here we focus on the
cytoskeleton regulatory mechanisms. Chicken line differences resulted in a higher expression in line B
compared to line A. Following Salmonella infection line A shows up regulation and line B shows some
down regulation of the pathway. The pathway shows equal expression between the lines after 24 h
infection with Salmonella. These results are consistent with the results described above.
WNT signalling: Contrary to the results described above this pathway shows many both up and down
regulated genes. Chicken line differences resulted in a higher expression in line B compared to line A
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for subpathways leading to stimulation of the cell cycle, which may be affected by the “Adherens
junction” pathway, and to Cytoskeletal changes, which may be affected by the “Focal adhesion”
pathway. Following Salmonella infection line A shows up regulation of both processes. No such effect
was observed in line B. After24 h infection with Salmonella the subpathways are still higher expressed
in line B compared to line A. Thus, the up regulation associated with Salmonella infection not sufficient
to level the line differences.
B-cell receptor signalling: The B-cell receptor pathway may be involved in the reaction of the chicken
to Salmonella infection, and one subpathway of this pathway leads to input for the “Regulation of
cytoskeleton” pathway. Chicken line differences resulted in a higher expression in line A compared to
line B of this latter subpathway. Following Salmonella infection line A shows down regulation and line
B shows up regulation of this subpathway. This subpathway shows equal expression between the lines
after 24 h infection with Salmonella. It can be concluded that this equal expression levels is the result
of the differential regulation of the lines.
T cell receptor signalling: Chicken line differences seem not to affect expression of this pathway.
Following Salmonella infection both lines show up regulation of several genes in this pathway, where
especially in line A more up regulated genes were found than in line B. Only in line A up regulation of
the input signal for the “Regulation of actin cytoskeleton” pathway is visible. This subpathway shows
equal expression between the lines after 24 h infection with Salmonella.
The Regulation of actin cytoskeleton pathway is in the centre of all these regulatory mechanisms. The
pathway describes the regulation of several different steps in changes in the actin cytoskeleton and
interactions with other pathways. Chicken line differences resulted in a higher expression in line B
compared to line A of the subpathways leading to the formation of stress fibres and two different
subpathways leading to input in the “Focal adhesion” pathway. Following Salmonella infection line A
shows up regulation of one subpathway leading to input in the Focal adhesion pathway (but not the
other) and uncertain regulation of stress fibre formation. Line B shows limited regulation. This
subpathway shows equal expression between the lines after 24 h infection with Salmonella.
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Network: Apoptosis mechanism
The Apoptosis pathway: Chicken line differences resulted in differential gene expression between the
chicken lines, but the direction seems uncertain. Following Salmonella infection both lines react to
Salmonella infection with induced expression of several genes of this pathway suggesting that
apoptosis is induced as a reaction to infection. This pathway shows differential expression between the
lines after 24 h infection with Salmonella including more genes showing lower expression in line B than
in line A, but no direction towards a specific effect could be observed.
The JAK-STAT signalling pathway is composed of several subpathways directing towards, cell cycle,
anti apoptosis, apoptosis, and proteolysis mechanisms. Chicken line differences resulted in some genes
in this pathway showing differential expression between the chicken lines but this seems not to be
directed towards a specific subpathway. Following Salmonella infection both lines show up regulation
of subpathways affecting cell growth and differentiation, and anti apoptosis. This pathway shows
higher expression in line A than in line B after 24 h infection with Salmonella. It can be concluded that
this differential expression levels is the result of a higher up regulation in line A than in line B.
The Natural Killer cell mediated cytotoxicity pathway affects both the apoptosis and the JAK-STAT
signalling pathway. Chicken line differences resulted in differential expression of genes in the pathway
but no clear effect on the “JAK-STAT pathway” could be found. Following Salmonella infection both
lines show up regulation of the subpathway involved in regulating input to the “JAK-STAT” pathway.
After 24 h infection with Salmonella infection the subpathway influencing the “JAK-STAT” pathway is
higher in line A than in line B. These results suggest that the up regulation is higher in line A than in
line B. The effect on the apoptosis pathway may be also affected by the up regulated release of
cytokines in line B. This may suggest that the chicken lines differ in the regulation of this pathway
following Salmonella infection.
The Transforming Growth Factor (TGF) beta pathway is involved in regulating proliferation,
differentiation, and apoptosis of tissues mainly of mesodermal origin. Chicken line differences resulted
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in a higher expression in line B compared to line A. Following Salmonella infection line A shows up
regulation of several genes in this pathway. None of the genes of the pathway in line B showed
regulation. This subpathway shows higher expression in line B compared to line A after 24 h infection
with Salmonella. This suggests that the up regulation of line A is not sufficient to equal the expressions
between the lines. The results suggest that the input for the MAPK pathway may be up regulated,
especially in line A.
The Mitogen-Activated Protein Kinase (MAPK) signalling pathway is involved in many regulatory
processes. Here we focus on apoptosis-related effects. Chicken line differences resulted in
contradictory expression differences between the chicken lines for the subpathway regulating
proliferation and differentiation of cells. Subpathways leading to apoptosis seem not to differ between
the lines. Following Salmonella infection both lines show up regulation of the input for the apoptosis
pathway although line B to a lesser extends than line A. Line A up regulate the subpathway for
stimulation of proliferation and differentiation of cells. However, after 24 h infection with Salmonella
infection the proliferation and differentiation affecting subpathway seems to be higher expressed in
line B than in line A. Subpathways leading to apoptosis seem not to differ between the lines.
Network: Regulation of energy metabolism
The Glycolysis / Gluconeogenesis pathway: Chicken line differences not affected expression of the
pathway. Following Salmonella infection the expression in line A is reduced in several steps. No such
effect could be observed in line B. After 24 h Salmonella infection line B shows higher expression than
line A. Thus, the difference in expression after infection is related to regulation in line A. The pathway
has several links to other pathways (both input to and from) including “Pentose phosphate” pathway,
”Glycerolipid” pathway, amino acid pathways, and nucleotide pathways, some of which seem to be
involved in regulation of energy metabolism too and will be reported and discussed below.
The Fatty acid metabolism pathway could be an alternative for the organism to supply energy to the
cells. Chicken line differences resulted in a higher expression in line B compared to line A. Following
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Salmonella infection both lines show down regulation of this pathway. After 24 h infection with
Salmonella this subpathway shows a higher expression in line B compared to line A. From these results
it cannot be concluded whether both lines react similarly or the reaction differs between the lines.
Although connections with other pathways exist these pathways seem not to affect the expression of
the “Fatty acid metabolism” pathway.
The Glycerolipid metabolism pathway has an output link towards the “Fatty acid metabolism” pathway.
Chicken line differences have not affected expression of this pathway. Following Salmonella infection
both lines show little regulation perhaps with a small exception for D-glyceraldehyde expression
regulation, which seem to be decreased in both lines similarly. This subpathway shows equal
expression between the lines after 24 h infection with Salmonella with the same exception. This may be
related to the effects found for the Glycolysis / Gluconeogenesis pathway, at least for line A.
The Pentose phosphate metabolism pathway: Chicken line differences have not affected expression of
this pathway. Following Salmonella infection line A shows some regulation of several genes of the
Glycolysis affecting subpathway. This subpathway shows equal expression between the lines after 24 h
infection with Salmonella.
The Purine metabolism pathway synthesized nucleotides and their precursors. The pathway delivers
input for the “Pentose phosphate metabolism” pathway. Chicken line differences resulted in a higher
expression in line B compared to line A. Following Salmonella infection both lines show up regulation
of several genes in this pathway. This subpathway shows higher expression in line B compared to line
A after 24 h infection with Salmonella. From the data it is not possible to conclude on differences
between the lines.
The Insulin signalling pathway may be suggested a regulatory pathway for energy metabolism. The
pathway has connections with other pathways including “Glycolysis / Gluconeogenesis”, “Fatty acid
metabolism”, and “Apoptosis”, and affect proliferation and differentiation. Chicken line differences
resulted in a higher expression in line B compared to line A of the subpathways leading to input for
“Glycolysis / Gluconeogenesis”. Following Salmonella infection both lines show some regulation but
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the data is too limited to direct this to a specific path or process. This subpathway shows equal
expression between the lines after 24 h infection with Salmonella.
Other Metabolic pathways
Three pathways involved in amino acid metabolism were found:
Val, Leu, Ile degradation: Chicken line differences resulted in a higher expression in line B compared to
line A. Following Salmonella infection this is reversed for the first part of the degradation pathways
until coupling with CoA. Both lines react similarly to infection: The first part of the degradation
pathways until coupling with CoA is up regulated, the latter part, which comprises the actual
degradation steps, is down regulated. The observed changes in the expression after 24 h infection with
Salmonella of the first part of the degradation pathways until coupling with CoA suggest that the up
regulation is much higher in line A than in line B. However, both lines reduce the final steps of amino
acid degradation.
The Gly, Ser, Thr metabolism pathway: Chicken line differences have limited effect on this pathway: one
small subpathway leading to Porphirine biosynthesis may be affected because line B shows higher
expression than line A. Following Salmonella infection this subpathway is down regulated in line B. The
subpathway shows equal expression between the lines after 24 h infection with Salmonella.
Limited information is available for the Beta-Alanine metabolism pathway. Chicken line differences in
the reaction to infection seem not to affect expression of the pathway. Therefore, input for other
pathways such as “Fatty acid biosynthesis”, “Arginine and proline metabolism”, “Pantothenate and CoA
biosynthesis”, and “Pyrimidine” synthesis are also unchanged.
Amino acid metabolism also involved breakdown of proteins. The proteasome is important for
protein breakdown. The proteasome is a complex molecule consisting of the products of at least 32
different genes. Chicken line differences may have affected expression of a few genes. Following
Salmonella infection line A may show some up and down regulation. This subpathway shows equal
expression between the lines after 24 h infection with Salmonella.
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The KEGG database search highlighted more general degradation pathways:
Caprolactam (C6H11ON, used for the synthesis of nylon 6) degradation:
Chicken line differences resulted in a higher expression in line B compared to line A. Following
Salmonella infection line A show down regulation. After 24 h infection with Salmonella a higher
expression in line B compared to line A was observed. These results suggest that the line difference is
enhanced by infection.
The Metabolism of Xenobiotics by cytochrome P450 pathway:
Chicken line differences showed no marked differences. Following Salmonella infection both lines
show sharp down regulation. But after 24 h infection with Salmonella this subpathway shows equal
expression between the lines.
Immune response pathways
Immune response pathways may act via multiple mechanisms. For the T-cell receptor signalling and
the B-cell receptor signalling pathways we refer to the section on regulation of actin cytoskeletal
changes above. The Cytokine – cytokine-receptor interaction pathway returned by the KEGG database
consists of many molecule type-specific interactions. Neither chicken line differences nor reaction to
infection seems to affect expression of the pathway. Contrary to this the Adipocytokine signalling
pathway: Chicken line differences resulted in a higher expression in line B compared to line A.
Following Salmonella infection line A shows up regulation of this pathway. No such effect was found
for line B. This subpathway shows equal expression between the lines after 24 h infection with
Salmonella.
The Gap-junction pathway: Neither chicken line differences nor reaction to infection seems to affect
expression of the pathway. Chicken line differences influenced the Cytokine-cytokine receptor
interactions with which the pathway starts.
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Many genes of the Complement and coagulation cascades pathway were present on the
microarray. However, neither chicken line differences nor reaction to infection seems to affect
expression of the pathway.
Other body functional pathways
The Androgen and estrogen metabolism pathway suggests that gender related mechanisms
may be involved. Chicken line differences resulted in a higher expression in line B compared to line A
of the Estradiol and Estradiol-17β metabolisms subpathway. Following Salmonella infection several
genes in line B show down regulation. No such effect was found in line A. This subpathway shows
higher expression in line B compared to line A after 24 h infection with Salmonella. Therefore, it can be
concluded that this down regulation is statistically significant, but not big enough to equal the
expression between the lines.
The Circadian rhythm pathway suggests involvement of day-night rhythm. Chicken line differences
resulted in a higher expression in line B compared to line A. Following Salmonella infection both lines
show down regulation of the pathway. After 24 h infection with Salmonella the expression is higher in
line B compared to line A.
Bile acids may be directly involved in the resorption of fatty acids by the intestine. The Bile acid
biosynthesis pathway: Chicken line differences resulted in a higher expression in line B compared to
line A. Following Salmonella infection line B show down regulation of several genes of this pathway.
No such effect was found in line A. After 24 h infection with Salmonella the expression is higher in line
B compared to line A. Thus we can conclude that the down regulation in line B is not sufficient to equal
the expression of the pathway between the lines.