Epithelial cell invasion by salmonella typhimurium induces modulation of genes controlled by aryl hydrocarbon receptor signaling and involved in extracellular matrix biogenesis

Figures & data

Table 1. Characteristics and designation of the strains used in this study.

Strains	Designation used in the article	References
Salmonella Typhimurium 14028	WT	ATCC
Salmonella Typhimurium 14028 express rck and pagN in culture	STM- Z T	This study
Salmonella Typhimurium 14028 express rck and pagN in culture and invalidated for invA	STM- Z	This study
Salmonella Typhimurium 14028 invalidated for rck, and pagN	STM- T	This study
Salmonella Typhimurium 14028 invalidated for invA, rck, and pagN	STM-3d	Roche et al 2018

Figure 1. Invasion of IEC-6 cells by S. Typhimurium strains entering cells by different entry pathways.

Using a gentamicin assay invasion capabilities of S. Typhimurium strains capable of entry by both trigger and zipper mechanisms (STM-ZT), by a trigger (STM-T) or zipper (STM-Z) entry mechanism, or by none of the known mechanisms (STM-3d) were compared. Bacteria were deposited on IEC-6 with a multiplicity of infection of 10, for 1.5 h, then gentamicin was added (100µg/ml) for 1.5 h. Intracellular bacteria levels were assessed after cell lysis. This result corresponds to the mean of 6 independent experiments. Statistical analyses using a Mann Whitney test were performed using GraphPad Prism version 6.07 for Windows. (Significance was *** at p<0.001, ** at p<0.01, * at p<0.05.

Table 2. Paired comparisons of gene expression modification induced in IEC-6 cells exposed to the S. Typhimurium strains STM- Z T, STM- Z, or STM- T.

STM-T/STM-Z
Gene Name	FC	GO biological process
XR_589495	11,6	nc RNA
XR_344993	3,7	nc RNA
Mlr1	-2,5	transcription factor RNA polymerase II specific
Dclk1	-2,6	neuronal apoptosis and neurogenesis
Nxf3	-2,6	polyA+ mRNA export
Smpdl3a	-3,0	nucleoside triphosphate catabolic process
Nxph4	-3,0	neuropeptide signalling pathway
Gc	-3,0	vitamin D metabolic process
XM_008775294	-3,9	nc RNA
Rhoh	-4,1	T cell differentiation, negative regulation of I-kappaB kinase/NF-kappaB signalling
Tgfbr1	-5,0	transforming growth factor beta receptor signalling pathway
STM-Z/STM- Z T
Gene Name	FC	GO biological process
Rhoh	4,7	T cell differentiation, negative regulation of I-kappaB kinase/NF-kappaB signalling
XM_008775294	3,6	nc RNA
Smpdl3a	2,7	nucleoside triphosphate catabolic process
Nxf3	2,6	polyA+ mRNA export
Gc	2,7	vitamin D metabolic process
STM-T/STM-Z T
Gene Name	FC	GO biological process
XR_589495	10	nc RNA
XR_344993	4,1	nc RNA

DE genes with a p value <0.05 are represented.

Figure 2. Specific and shared differentially expressed genes between IEC-6 cells exposed to STM-ZT, STM-Z or STM-T strains compared to non-exposed cells (cont).

Venn diagram showing the number of specific and shared differentially expressed (DE) genes (exposed/non-exposed cells) obtained after a whole-expression genomic profiling of IEC-6 cells infected by STM-ZT, STM-Z or STM-T strains. Gene expression analyzes were performed after 1.5 h of interaction between IEC-6 and STM strains and 1.5 h of gentamicin to kill extracellular bacteria. DE genes with a p value <0.05, and a fold change (FC) > |2| are represented.

Figure 3. Functional enrichment analysis of biological processes.

Analysis of differentially expressed (DE) genes (exposed/non-exposed cells) obtained after a whole-expression genomic profiling of IEC-6 cells infected by STM-ZT, STM-Z or STM-T strains. Gene expression analyses were performed after 1.5 h of interaction between IEC-6 and STM strains and 1.5 h of gentamicin to kill extracellular bacteria. Clustering heatmap plot of functional sets of gene ontology (GO) terms was obtained using ViSEAGO showing the major biological processes. The plot combines a dendrogram based on Wang’s semantic similarity distance and ward.D2 aggregation criterion, a heatmap of -log₁₀(p-value) from functional enrichment tests and information content (IC). Focus is made on several major biological processes. For the sake of clarity, we only kept the genes showing high levels of expression |log₁₀(FC)| > 5).

Table 3. Differentially expressed genes in the microarray experiment associated with stimulation or inhibition of the ECM.

Gene	ID	FC	Functional relevance with regard to ECM
Genes associated with urokinase pathway
SerpinB2	NM_021696	24.8	Inhibition urokinase plasminogen activator (uPA) {Tang, 2013 #2267}
Serpine1	NM_012620	6.1	Inhibition of uPA {Tang, 2013 #2267}
Cyr61	NM_031327	2.0	Enhances Timp1 and Serpine1 production {Chen, 2001 #2282}
Spry1	NM_001106427	2.3	Decreases expression of uPAR at cell surface
Timp1	NM_053819	2.6	Inhibition of MMP {Brew, 2000 #2299}
Plaur	NM_134352	2.3	Receptor urokinase (uPAR) {Mekkawy, 2014 #3033} {Liu, 2014 #2281}
Genes involved in ECM component synthesis/degradation
Sod2	NM_017051	7.8	Protection of heparan and type I collagen {Petersen, 2004 #2288}
Has1	NM_172323	6.4	Biosynthesis of hyaluronan
Mt2A	NM_001137564	4.0	Upregulation of collagenase expression
Ugdh	NM_031325	3.4	Biosynthesis of glycosaminoglycans
Lrp4	NM_031322	3.2	Induction of ECM genes expression {Asai, 2014 #2289}
Uap1	NM_017259	2.2	Biosynthesis of N-glycans
Adamts4	NM_023959	2.1	Aggrecan degradation {Westling, 2002 #2315}
Genes involved in ECM formation
Plet1	NM_001014209	13.5	Tissue repair {Zepp, 2017 #2298}
Tnfaip6	NM_053382	3.3	Involved in ECM stability {Lauer, 2013 #2844}
Creb3l1	NM_001005562	2.0	Assembly ECM
Lmo7	NM_001001515	-4.1	Negative regulator of ECM deposition {Xie, 2019 #2847}
Fgf18	NM_019199	-3.8	Positive regulator of ECM deposition
Genes involved in fibrosis
Tnf	NM_01267	23.3	Pro- anti- fibrotic, contradictory observations {Distler, 2008 #3284}
Tnfsf15	NM_145765	4.5	Pro-fibrotic {Barrett, 2012 #2269}
Arel1	NM_001106744	2.2	Pro-fibrotic {Lear, 2016 #2275}
Wnt10A	NM_001108227	2.8	Pro-fibrotic {Oda, 2016 #2273}
Antxr2	XM_008764602	2.8	Pro-fibrotic {Burgi, 2017 #2276}
Tgfb3	NM_013174	-3.3	Pro-fibrotic {Guo, 2021 #3027}
Fst	NM_012561	4.1	Anti-fibrotic {Patella, 2006 #2317}

Differentially expressed (DE) genes with a p value <0.05, and a fold change (FC) > |2| are represented. FC represents the ratio of gene expression between IEC-6 cells exposed to S. Typhimurium strain STM-Z T and non-exposed cells.

Table 4. Differentially expressed genes included in the IPA upstream regulator analysis that may be activated by the AHR pathway.

Predicted up-regulations		Predicted down-regulations		Inconsistent findings		No prediction
Gene name	Functional category	Gene name	Functional category	Gene name	Functional category	Gene name	Functional category
ACKR3	G-protein Coupled Receptor	ADAM19	Peptidase	C1QB	Other	ALDH3A1*	Enzyme
ACTN1	Transcription regulator	ADAMTS5	Peptidase	CDC25C	Phosphatase	ATOH8	Transcription regulator
ADM	Other	CDKN2C	Transcription regulator	CDH1	Other	BTG2	Transcription regulator
AHRR	Transcription regulator	HEY2	Transcription regulator	CDKN1A	Kinase	Casp8	Peptidase
ALDOA	Enzyme	TGFB2	Growth factor	CEBPA	Transcription regulator	CCNG2	Other
AREG	Growth factor			CERS4	Transcription regulator	CDH7	Enzyme
ARG2	Enzyme			E2F1	Transcription regulator	COL27A1	Other
CCL20	Cytokine			E2F8*	Transcription regulator	CYP2S1	Enzyme
CCL5	Cytokine			FBN2	Other	EBF1	Transcription regulator
CD274	Enzyme			FN1	Enzyme	FAS	Transmembrane receptor
CD3D	Transmembrane receptor			FOSL1	Transcription regulator	GHITM	Other
CERS6*	Transcription regulator			GADD45A	Other	HECTD2	Enzyme
CXCL10	Cytokine			GAS1	Other	JAG1	Cytokine
CXCL2	Cytokine			HES1	Transcription regulator	Mt1	Other
CYP1A1	Enzyme			HIF1A	Transcription regulator	NTM2	Enzyme
CYP1B1	Enzyme			HP90AA1	Enzyme	NNMT	Enyme
CYP2C8	Enzyme			HSPA5	Enzyme	TNFAIP8L1	Other
CCL2	Cytokine			JUN	Transcription regulator	VEGFA	Growth factor
Cdc42	Enzyme			JUNB	Transcription regulator	VEGFC	Growth factor
EDN1	Cytokine			LBP	Transporter
EGLN3	Cytokine			PLAT	Peptidase
FMO3	Enzyme			PLK1	Kinase
HK2	Kinase			Pcp4I1	Other
IL1R1	Transmembrane receptor			SOX2	Transcription regulator
IL1R2	Transmembrane receptor			STEAP2	Enzyme
INSIG2	Other			Saa3	Other
IRF1	Transcription regulator			TNF	Cytokine
ITGA5	Transcription regulator			VIM	Other
KDSR	Enzyme
MYC	Transcription regulator
NFE2L2	Transcription regulator
NOTCH	Transcription regulator
NQO1	Enzyme
PDE4B	Enzyme
PTGS2*	Enzyme
PTX3	Other
SDC1	Enzyme
SERPINB2	Other
SERPINE1	Other
SLAMF8	Other
SLC2A1	Transporter
SOCS3	Phosphatase
SOD2	Enzyme
SPTLC2	Enzyme
THBS1*	Other
TIPARP*	Enzyme
UGCG	Enzyme
UGT1A6	Enzyme

The genes are clustered into four categories, which correspond to specific link colours in the Figure S1: “predicted up-regulations” (orange links), “predicted down-regulations” (blue), “inconsistent findings” (yellow) and “no prediction” (grey). The genes are represented in different colours corresponding to the direction of variation (see also Figure S1): strongly up-regulated genes are in red, lightly up-regulated genes are in pink, strongly down-regulated genes are in dark green, and lightly down-regulated genes are in light green. The functional categories of the genes (actually corresponding to different shapes in Figure S1) are also reported in the table.

Table 5. Comparison of gene expression determined by microarray and qRT PC.

Gene	FC qRT PCR	FC microarray
Adamts4	2.7	2.1
Ajuba	-2.4	-2.2
Angptl4	6.4	11.3
Areg	3.1	2.0
Arl4c	-2.2	-2.1
Bdkrb1	4.8	2.5
Bmp4	-5.3	-3.2
Cd302	2.2	2.2
Cd44	2.4	2.0
Ceacam	1.4§	2.0
Creb3l1	2.3	2.0
Cyr61	2.4	2.0
Ephb3	-4.0	-2.2
Errfi1	2.3	2.0
Ets2	2.8	2.5
Fat1	1.9	2.0
Fgf18	-3.8	-3.8
Flrt3	-2.2	-2.2
Gabarapl1	2.3	2.4
Gja1	2.2	2.0
Icam1	6.5	6.5
Lpar1	2.1	2.0
Nrg1	3.6	3.8
Olr1	5.7	5.0
Pla2g2a	1.8	2.0
Plaur	1.1§	2.3
Procr	2.5	2.0
Ptpn12	2.4	3.1
Rhbdf2	4.1	3.3
Rtp4	5.8§	2.2
Sdc4	2.5	2.2
Serpinb2	23	24.8
Serpine1	7.2	6.1
Sfrp2	4.6	3.6
Snx18	1.5§	2.3
Timp1	3.1	2.6
Tnfaip6	4.0	3.3
Tnfrsf11b	5.7	5.1
Tnfsf15	12	4.5
Vegfa	2.4§	2.0

Fold changes (FC) represents the ratio of gene expression between IEC-6 cells exposed to STM-Z T and non-exposed cells. FC with a § have a p value >0.05, FC without § have a p value <0.05.

Figure 4. Invasion of the STM-ZT and STM-3d strains impaired for entry by pharmaceutical means.

Invasion ability of the STM-ZT and STM-3d was analyzed using a gentamicin protection assays in the presence of 1 mM amiloride (Am) and 10 µg/mL chlorpromazine (CPZ). IEC-6 cells were pre-treated for 30 min with the drugs, or with the appropriate solvent used for the dilution of the drugs as control. Infection was performed in the presence of the drugs; bacteria were deposited on IEC-6 with a multiplicity of infection of 10, for 1.5 h, then gentamicin was added (100µg/ml) for 1.5h. Intracellular bacteria levels were assessed after cell lysis. This result corresponds to the mean of five independent experiments. Relative invasion corresponds to the number of internalized bacteria in cells treated relative to the non-treated cells infected by STM-ZT arbitrarily set at 100%.

Table 6. Expression profile of selected ECM-associated genes in IEC-6 cells treated or not with chlorpromazine and amiloride before and during infection with the STM-Z T strain.

Gene	STM-Z T/Cont	Ch+Am/Cont
SerpinB2	37	6.5
Angptl4	14	7.2
Icam1	8.6	4.7
Cyr61	4.2
Rtp4	9.2
Tnfsf15	50
Vegfa	4.2
Ephb3	-4.1
Olr1	9.2
Rhbdf2	9.2
Serpine1	8.4
Tnfrsf11b	6.0
Tnfaip6	5.8
Ceacam1	4.9
Bdkrb1	4.8
Isg15	4.0
Ptpn12	3.5
Timp1	3.5
Adamts4	3.3
Ets2	3.1
Nrg1	3.0	2.0
Areg	2.8
Snx18		2.8
Procr	2.7
Cd302	2.5
Gja1	2.3
Creb3l1	2.2
Plaur	2.2
Fat1		2.2
Sdc4	2.1
Bmp4	-2.2
Fgf18	-3.2

Figure 5. Western blotting analysis of IEC6 cells infected by the different S. Typhimurium strains.

Alteration of Extracellular Matrix Proteins (ECM) after bacterial invasion is illustrated by western blotting analysis of laminin and fibronectin. Bacteria were deposited on IEC-6 with a multiplicity of infection of 10, for 1.5 h, followed by gentamicin (100µg/ml) for 1.5 h. Cells were then resuspended in Laemmli buffer and denaturated 10 min at 100°C. Samples from Non-infected cells (NI) and cells infected by STM-ZT, STM-Z, STM-T or STM-3d strains were loaded in a 4-15% Miniprotean TGX Precast Protein gels and then transferred to a nitrocellulose membrane followed by detection of laminin (A) and fibronectin (B). Protein concentrations were normalized to the tubulin-blotting reference and the ratios were expressed relative to non-infected cells. The Regions of interest (ROI) taking into account for this normalization are represented by the rectangles.

Data availability statement

The data that support the findings of this study are available in the GEO database with the accession number GSE151881.