Julien Delmas
email: julien.delmas@uca.fr

Lucie Gibold

Tiphanie Faïs

Sylvine Batista

Martin Leremboure

Clara Sinel

Emilie Vazeille

Vincent Cattoir

Anthony Buisson

Nicolas Barnich

Guillaume Dalmasso

Richard Bonnet

Metabolic adaptation of adherent-invasive Escherichia coli to exposure to bile salts

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The adherent-invasive Escherichia coli (AIEC) pathogroup was initially characterized in isolates from the ileal mucosa of Crohn's disease (CD) patients [START_REF] Martin | Enhanced Escherichia coli adherence and invasion in Crohn's disease and colon cancer[END_REF][START_REF] Darfeuille-Michaud | High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease[END_REF][START_REF] Kotlowski | High prevalence of Escherichia coli belonging to the B2+D phylogenetic group in inflammatory bowel disease[END_REF][START_REF] Baumgart | Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum[END_REF][5] . These bacteria strongly adhere to and invade intestinal epithelial cells (IECs), survive within macrophages, move into the deep tissues and activate immune cells to induce inflammatory cytokine secretion [START_REF] Darfeuille-Michaud | Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease[END_REF][START_REF] Glasser | Adherent Invasive Escherichia coli Strains from Patients with Crohn's Disease Survive and Replicate within Macrophages without Inducing Host Cell Death[END_REF][START_REF] Eaves-Pyles | Escherichia coli isolated from a Crohn's disease patient adheres, invades, and induces inflammatory responses in polarized intestinal epithelial cells[END_REF] .

Several studies have identified genes that are important for AIEC ileal colonization: type 1 pili and flagella facilitate binding to and invasion of the epithelial cell; long polar fimbriae (LPF) aids in the binding of these bacteria to M cells overlying Peyer's patches; Vat-AIEC protease promotes mucins degradation. The expression of most these virulence factors is modulated by bile salts, suggesting that bile salts may be used by AIEC as an environmental signal to promote their colonization [START_REF] Chassaing | Bile salts induce long polar fimbriae expression favouring Crohn's disease-associated adherent-invasive Escherichia coli interaction with Peyer's patches[END_REF][START_REF] Gibold | The Vat-AIEC protease promotes crossing of the intestinal mucus layer by Crohn's disease-associated Escherichia coli[END_REF][START_REF] Vazeille | GipA Factor Supports Colonization of Peyer's Patches by Crohn's Disease-associated Escherichia Coli[END_REF][START_REF] Sevrin | Adaptation of adherent-invasive E. coli to gut environment: Impact on flagellum expression and bacterial colonization ability[END_REF] . Other genes identified to date as required for colonization of AIEC are involved in the acquisition and metabolism of essential nutrients [START_REF] Dogan | Inflammation-associated adherent-invasive Escherichia coli are enriched in pathways for use of propanediol and iron and M-cell translocation[END_REF] . In recent years, a huge body of literature has provided evidence that most enteropathogens are equipped with a large set of specific metabolic pathways to overcome nutritional limitations in vivo, thus increasing bacterial fitness during infection [START_REF] Staib | From food to cell: nutrient exploitation strategies of enteropathogens[END_REF] . These adaptations include the degradation of myo-inositol, ethanolamine cleaved from phospholipids, fucose derived from mucosal glycoconjugates, 1,2propanediol as the fermentation product of fucose or rhamnose and several other metabolites not accessible for commensal bacteria or present in competition-free microenvironments.

Therefore, deciphering the metabolic requirements of AIEC is central to the understanding of their ability to colonize a host. In addition, little information is available regarding the effects of bile salts on E. coli metabolism. In the present study, we showed that secondary metabolic pathways in AIEC are modulated by bile salts, providing them with energy as well as carbon and nitrogen sources to colonize the intestinal mucosa.

Results

Bile salts altered expression of metabolic genes in LF82

For transcriptome analysis, an AIEC strain (LF82) isolated from a chronic ileal lesion of a patient with CD was grown for 24 h in the presence or absence of 1% bile salts [START_REF] Darfeuille-Michaud | Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease[END_REF] . The analysis of data showed a profound effect of bile salts with 1796 differentially expressed genes (DEG), including 1138 genes that showed an increase and 658 genes that showed a decrease in mRNA abundance, representing approximately 40% of the genome (additional file 1: Methods, Fig. S1,S2). Among these DEGs, 517 genes (29%) encoded proteins of unknown function.

After classification into functional categories, we observed 255 DEGs among the 722 genes involved in metabolism (35%), including 182 up-regulated and 73 down-regulated genes. In the presence of bile salts, the expression of genes encoding proteins involved in the degradation, utilization and assimilation of compounds was significantly induced relative to the other overexpressed genes involved in metabolism (Fig. 1A). Conversely, bile salts induced a repression of genes involved in biosynthesis relative to the other underexpressed genes involved in metabolism. More particularly, the expression of genes involved in the degradation of amines, alcohols, carboxylates and in secondary metabolism were overexpressed for 47% of them (Fig. 1B). Thus, bile salts induce a global up-regulation of genes involved in degradation.

We identified the most highly expressed genes, i.e., those showing an increase of at least 4-fold (i.e., log2 = 2) in mRNA abundance in bacteria grown with bile salts in comparison to bacteria grown without bile salts. These most up-regulated genes encoded proteins involved in sugar degradation (rhamnose, arabinose, galactose and mannose), L-ascorbate degradation, amino acid degradation (tryptophane, glutamine and arginine), citrate degradation, the methyl-citrate pathway and ethanolamine utilization. Ethanolamine (EA) can be used as a carbon and/or nitrogen source by many bacterial species and provide acetyl-CoA. Citrate degradation and the methyl citrate pathway provide acetate and pyruvate, respectively. Since acetyl-coA, acetate and pyruvate are central metabolic intermediates, we focused on these three pathways (additional file 1: Fig. S3).

Induction of ethanolamine metabolism by bile salts

The genetic information required for the degradation and utilization of EA by E. coli is encoded by 17 genes in the eut operon. In the presence of bile salts, all of these genes were significantly up-regulated in AIEC LF82 (2.5 to 12.2-fold, Table 1). The intestinal tract provides a rich source of EA in the form of phosphatidylethanolamine due to its presence in bacterial and eukaryotic cell membranes and in the host diet. To evaluate the ability of the AIEC LF82 strain to utilize EA as a nitrogen source, EA was added along with 0.1% glucose to mM9 minimal medium supplemented with bile salts (mM9b). To test the ability of the bacterium to use EA as a carbon source, EA and NH4Cl were added to mM9b. As shown in Fig. 2, the AIEC LF82 strain failed to grow in minimal medium supplemented with EA and NH4Cl. This finding demonstrated that the LF82 strain is unable to utilize EA as a carbon source but can use EA as the sole nitrogen source. Ethanolamine ammonia lyase, encoded by the genes eutB and eutC, converts EA to ammonia and acetaldehyde (additional file 1: Fig. S3). While ammonia can serve as a cellular source of reduced nitrogen, acetaldehyde is further converted to acetylcoenzyme A by an aldehyde oxidoreductase encoded by eutE and enters the carbon pool of the cell. As shown in Fig. 2, the mutant defective for EutB (LF82eutB) was unable to grow in mM9b medium with EA as the sole nitrogen source, while complementation in trans with the eutB gene (LF82eutB/peutB) restored bacterial growth. In contrast, the LF82eutE mutant showed a growth phenotype similar to the LF82 strain. These results clearly demonstrated that the AIEC strain LF82 can use EA as a nitrogen source and that a functional ethanolamine ammonia lyase including EutB is required for the release of ammonia.

Utilization of ethanolamine confers a competitive advantage to AIEC strains

The eut operon is conserved within E. coli species. In order to determine whether the utilization of EA in presence of bile salts is a common trait shared by AIEC strains, we studied a collection of Escherichia coli isolates including AIEC (n=18) and non-AIEC strains (n=18).

Non-AIEC strains grew more slowly in mM9b minimal medium supplemented with glucose (0.01%) and EA than AIEC strains (p=0.009, Fig. 3A, additional file 1: Fig. S4). Nevertheless, growth was similar between group strains in mM9b minimal medium supplemented with glucose (0.1%) and NH4Cl (Fig. 3B, additional file 1: Fig. S4). Thus, ethanolamine metabolism could be a trait associated with AIEC strains. The growth of the AIEC LF82 strain was high in comparison to those of the E. coli strain MG1655 in mM9b-EA (Fig. 4A; additional file 1: Fig. S5). To explain the difference in growth, the ratio of the mRNA level of several genes of the eut operon was measured in both strains during the early phase of growth. The eut genes were found to be significantly up-regulated in AIEC strain LF82 when bile salts were added to the culture medium (Fig. 4B), and significantly up-regulated in comparison to the MG1655 strain (Fig. 4C). These data suggest that AIEC strain LF82 degrades EA more efficiently than other E. coli in the presence of bile salts. The utilization of EA could provide a nutritional advantage for AIEC. To test this hypothesis, competitive assays were performed between LF82 strain, its isogenic eutB mutant and the strain MG1655. The LF82 strain was able to grow in the presence of EA much more effectively than its isogenic mutant or the MG1655 strain (Fig. 4D). Interestingly, competitive index values close to 1 were obtained when the LF82eutB and MG1655 strains were co-incubated, indicating that EA present in mM9b-EA was poorly used by the E. coli strain MG1655. Enterococci are prominent members of the normal human intestinal microflora. To analyze the capacity of Enterococcus to catabolize EA, 4 strains (Enterococcus faecium and three strains of Enterococcus faecalis) were incubated in mM9-EA with bile salts, and no growth was observed while they grow in mM9 supplemented with glucose and NH4Cl (data not shown). Altogether, these data suggest that, in the presence of bile salts, EA might be a privileged substrate for AIEC.

We next evaluated the capacity of AIEC LF82 to use EA to colonize the gut. Mice, with colitis caused by dextran sulfate sodium (DSS), were orally challenged with an equal mixture of the LF82 strain and the LF82eutB isogenic mutant. Quantification of bacteria in stool samples and ileal and colonic tissues revealed approximatively a one-log reduction in the LF82eutB-AIEC mutant compared with wild-type LF82 (Fig. 5A andB). Analysis of the competitive index of LF82 bacteria growth compared with isogenic mutant growth revealed a median competitive index of 35, 18 and 16 for stool, ileum and colon, respectively (Fig. 5C).

These results indicate that AIEC may gain a competitive advantage in the intestine by using EA.

An increased intracellular acetyl-CoA pool was induced by bile salts

Acetyl-CoA can be produced by EA degradation. EA is converted to ammonia and acetaldehyde, which is converted to acetyl-CoA by EutE, a component that is significantly increased in the presence of bile salts (RNA-seq data: log2 = 10.4; Table 1). Inside bacteria, acetyl-CoA is central to energy generation and to several biosynthetic pathways that utilize the activated two-carbon acetyl unit. However, EA is not the exclusive source of acetyl-CoA generation. Through its oxidative decarboxylation, pyruvate is a major source of acetyl-CoA.

Pyruvate is usually formed by glycolysis to be oxidized by the the tricarboxylic acid (TCA) cycle. Genes involved in glycolysis and pyruvate decarboxylation (aceE, aceF and lpd) were not up-regulated during exposure to bile salts (Table 2). Phophoenolpyruvate, which is the precursor of pyruvate in glycolysis, was present in the same amounts in bacterial cultures with or without bile salts (30.3 ± 2.1 and 29.2 ± 4.9 mg/L, respectively; p = 0.886), confirming that the glycolysis pathway was not up-regulated by bile salts. Acetate, for example, could also lead to the production of acetyl-CoA. Interestingly, genes encoding citrate lyase (citD, citE and citF) were up-regulated under bile conditions (Table 3). The citrate lyase converts citrate to oxaloacetate and acetate (Fig. S3). One of two distinct pathways by which E. coli activates acetate to acetyl-CoA is the activation of acetyl-CoA synthetase (Acs) [START_REF] Wolfe | The Acetate Switch[END_REF] . The acs gene was significantly up-regulated in the presence of bile salts (fold change: 2.6). Up-regulation of eutE and acs genes suggested that AIEC strain LF82 is able to produce a pool of acetyl-CoA under bile stress.

In order to test this hypothesis, the intracellular acetyl-CoA concentration was measured from bacterial cultures. The intracellular acetyl-CoA pool of LF82 strain increased when bile salts were added (5.2 ± 0.6 to 17.5 ± 4.3 mg/L; p = 0.0286) but not in mutants defective for ethanolamine ammonia lyase (LF82eutB) or deficient for citrate lyase (LF82citF) (4.1 ± 0.3 and 5.9 ± 0.5 mg/L, respectively) (Fig. 6).

These observations confirmed that EA and citrate degradation pathways activated upon bile salts exposition lead to acetyl-CoA production by the AIEC strain LF82. In contrast, the intracellular acetyl-CoA pool of the strain MG1655 slightly decreased in the presence of bile salts (7.2 ± 0.8 to 4.3 ± 0.8 mg/L). The eutB and citF genes were found to be significantly downregulated in the strain MG1655 grown in the presence of bile salts (12.5 and 4.2-fold, respectively; p = 0.0286). These data reinforce the fact that this strain and the AIEC strain LF82 have a different metabolism in the presence of bile salts.

Induction of 1,2-propanediol degradation and methyl-citrate pathway by bile salts

The results of the RNA-seq analysis showed that the entire group of genes (pdu operon) was induced in the presence of bile salts (Table 4) suggesting the formation of propionyl-CoA from 1,2-propanediol (Fig. S3). Interrestingly, pdu operon is significantly correlated with the AIEC pathotype [START_REF] Dogan | Inflammation-associated adherent-invasive Escherichia coli are enriched in pathways for use of propanediol and iron and M-cell translocation[END_REF] . E. coli is known to ferment fucose and rhamnose to 1,2-propanediol [START_REF] Boronat | Metabolism of L-fucose and L-rhamnose in Escherichia coli: differences in induction of propanediol oxidoreductase[END_REF] . Genes involved in the catabolism of rhamnose were up-regulated in the presence of bile salts (Table 4). Propionyl-CoA can also derive from propionate. Interestingly, the prpE gene encoding the enzyme that converts propionate to propionyl-CoA [START_REF] Wolfe | The Acetate Switch[END_REF] was significantly up-regulated by 9.8fold (Table 3). The propionyl-CoA is the precursor of 2-methylcitrate, the coactivator needed for transcriptional activation of the prpBCDE operon by the PrpR activator protein. RNA-seq data analysis revealed that genes involved in the methyl-citrate pathway (prpB, prpC, prpD, prpE, prpR) were strongly up-regulated (2.3 to 23.6-fold), with the exception of acnB, a gene that is constitutively expressed (Table 3). The methyl-citrate cycle converts propionyl-CoA to pyruvate and succinate (Fig. S3). Thus, pyruvate might be produced from 1,2-propanediol via a pathway that is activated under bile conditions in AIEC strain LF82.

Interactions between metabolic pathways

To obtain 2-methylcitrate and subsequently pyruvate, propionyl-CoA must be condensed with oxaloacetate by the PrpC enzyme (Fig. S3). Interestingly, prpC mRNA exhibited a 12.3fold increase in LF82 upon bile salt exposure. Oxaloacetate may have several origins, including the oxidation of L-malate. However, the gene encoding malate dehydrogenase was not deregulated in the presence of bile salts. Oxaloacetate is also derived from tartrate and citrate fermentation. RNA-seq analysis revealed that genes involved in those fermentations were induced in the presence of bile salts (Table 3). Interestingly, the intracellular oxaloacetate concentration of the LF82 strain significantly increased when bile salts were added to the culture medium (1.4 to 41.9 mg/L; p = 0.029), showing that oxaloacetate was available in the cell. Thus, bile salts may favor concomitant activation of (i) tartrate and citrate pathways, leading to the formation of oxaloacetate, (ii) rhamnose, propionate and 1,2-propanediol degradation, leading to the formation of propionyl-coA and subsequently the (iii) methylcitrate pathway leading to pyruvate (Fig. S6). Pyruvate can be transformed to acetyl-CoA by formate acetyltransferase II. Since pflD encoding formate acetyltransferase II was up-regulated 4.5-fold, pyruvate may also contribute to the increased intracellular acetyl-CoA pool under bile conditions. Thus, our results suggest that, in the presence of bile salts, these pathways act together to favor the formation of acetyl-CoA and to provide an energetic advantage to AIEC LF82 strain (Fig. S6).

Alternative metabolic pathways of LF82 strain in mice in the presence of bile salts

Our RNA-seq data clearly indicate that bile salts activate in vitro secondary metabolic pathways in AIEC strain LF82. As a test to elucidate whether bile salts are an in vivo metabolic activator of EA and 1,2 propanediol utilization and citrate and methyl-citrate pathways, the bile acid sequestrant cholestyramine was administred to C57BL/6J mice. Cholestyramine decreases bile-acid pool size and is associated with decreased Ibabp and Shp levels in the ileum [START_REF] Kong | Mechanism of Tissue-specific Farnesoid X Receptor in Suppressing the Expression of Genes in Bile-acid Synthesis in Mice[END_REF][START_REF] Lam | Bile acids inhibit duodenal secretin expression via orphan nuclear receptor small heterodimer partner (SHP)[END_REF] . Down-regulation of these host genes in the group treated with cholestyramine indicates that the treatment was efficient (additional file 1: Fig. S7). The levels of bacterial mRNA extracted from the ileum of untreated mice were significantly increased in comparison with those treated with cholestyramine (Fig. 7). This observation strengthens the hypothesis that bile salts in the small intestine alter the metabolism of AIEC strain LF82 by inducing alternative metabolic pathways.

Discussion

In this study, global RNA sequencing (RNA-seq) of AIEC reference strain LF82 has been used to decipher the impact of bile salts on the expression of metabolic bacterial genes. The results revealed a global up-regulation of genes involved in degradation and a down-regulation of those implicated in several biosynthetic pathways. The following four pathways were most noteworthy: ethanolamine utilization, citrate and 1-2 propanediol degradation, and methylcitrate pathway. Importantly, in LF82 bacteria isolated from the ileal mucosa of infected mice, the mRNA level of genes involved in these pathways were increased in comparison to mice treated with cholestyramine resin, which is a bile acid sequestrant, suggesting that these pathways are activated in vivo under bile conditions. Our results indicate that activation of these secondary metabolic pathways in LF82 AIEC favor the formation of acetyl-CoA (Fig. S6).

Acetyl-CoA is a potential energy source because it can be converted to acetyl-PO4 2-. The EutD phosphotransacetylase, for which the gene was up-regulated upon exposure to bile salts, can modify acetyl-CoA into acetyl-PO4 2-. This enzyme is a more efficient phosphotransacetylase than E. coli Pta, which has activity linked to acetate excretion and to the operation of the Tricarboxylic Acid cycle [START_REF] Bologna | Characterization of Escherichia coli EutD: a phosphotransacetylase of the ethanolamine operon[END_REF] . Thus, the present study provides insight into the pathways favored by AIEC LF82 for optimal energy generation.

Comparative genomic analysis of AIEC strains from different collections (Europe, North America and Australia) show that this pathobiont does not harbour an exclusive molecular signature [START_REF] Sepehri | Phylogenetic analysis of inflammatory bowel disease associated Escherichia coli and the fimH virulence determinant[END_REF][START_REF] Nash | Genome sequence of adherent-invasive Escherichia coli and comparative genomic analysis with other E. coli pathotypes[END_REF] , suggesting that AIEC strains have evolved from commensal E. coli by different mechanisms to favour their implantation in genetically susceptible CD patients. Our results identified two pathways, ethanolamine utilization and 1-2 propanediol degradation, that may be associated with the metabolic adaptation of the AIEC strains to their environments. The propanediol utilization (pdu) operon, which is prevalent among enteropathogenic bacteria such as Salmonella, Yersinia, Listeria, and Clostridium perfringens, is significantly correlated with the AIEC pathobiont [START_REF] Lawhon | Global regulation by CsrA in Salmonella typhimurium[END_REF][START_REF] Korbel | Systematic Association of Genes to Phenotypes by Genome and Literature Mining[END_REF][START_REF] Thomson | The Complete Genome Sequence and Comparative Genome Analysis of the High Pathogenicity Yersinia enterocolitica Strain 8081[END_REF] . In AIEC, the presence of pduC, which is significantly up-regulated in the presence of bile salts in our study, has been correlated with increased cellular invasion and bacterial persistence [START_REF] Dogan | Inflammation-associated adherent-invasive Escherichia coli are enriched in pathways for use of propanediol and iron and M-cell translocation[END_REF] . The utilization of 1,2-propanediol is therefore associated with virulent strains, and its utilization in this redirected metabolism in AIEC strain LF82 strengthens the hypothesis that this compound is important for the virulence of bacteria. Interrestingly, we demonstrated that ethanolamine can be used as a source of nitrogen and acetyl-CoA by the LF82 AIEC and confers a growth advantage to AIEC strains. It has been shown in the same way that the pathogenic E. coli O157:H7 strain EDL933 uses EA as a nitrogen source in the bovine intestine, whereas EA is poorly metabolized by the endogenous microbiota of the bovine small intestine, including commensal E. coli [START_REF] Bertin | Enterohaemorrhagic Escherichia coli gains a competitive advantage by using ethanolamine as a nitrogen source in the bovine intestinal content[END_REF] . EA is abundant in the intestinal tract due to the turnover and exfoliation of enterocytes as well as the high concentration of bacterial cells [START_REF] Garsin | Ethanolamine Utilization in Bacterial Pathogens: Roles and Regulation[END_REF] .

However, EA does not contribute to the colonization of all gut bacteria. EA catabolism in Enterococcus facecalis modestly reduces gut colonization efficiency [START_REF] Kaval | Loss of Ethanolamine Utilization in Enterococcus faecalis Increases Gastrointestinal Tract Colonization[END_REF] . The ability of AIEC to utilize EA might therefore provide them with a competitive advantage to successfully colonize the intestinal mucosa as observed in the present study with LF82 in a mouse colitis model.

Recently, it has been shown that uropathogenic Escherichia coli (UPEC) ethanolamine metabolism is required for effective bladder colonization in mice and that UPEC upregulates the expression of ethanolamine utilization genes during uncomplicated UTIs in humans [START_REF] Sintsova | Role of Ethanolamine Utilization Genes in Host Colonization during Urinary Tract Infection[END_REF] . Thus, we can speculate that optimization of the use of EA might be a metabolic adaptation of pathogenic E. coli.

EA is not only important for nitrogen metabolism but that it is also used as a signaling molecule in cell-to-cell signaling to activate virulence gene expression in EHEC [START_REF] Kendall | Ethanolamine Controls Expression of Genes Encoding Components Involved in Interkingdom Signaling and Virulence in Enterohemorrhagic Escherichia coli O157:H7[END_REF][START_REF] Gonyar | Ethanolamine and choline promote expression of putative and characterized fimbriae in enterohemorrhagic Escherichia coli O157:H7[END_REF] . The eut operon encodes the transcriptional regulator EutR that was found to bind promotors and directly activate the expression of the locus of enterocyte effacement (LEE) in EHEC [START_REF] Luzader | EutR is a direct regulator of genes that contribute to metabolism and virulence in enterohemorrhagic Escherichia coli O157:H7[END_REF] . Interrestingly, EA promotes also expression of fimbriae in EHEC, in particular of long polar fimbriae (LPF) [START_REF] Gonyar | Ethanolamine and choline promote expression of putative and characterized fimbriae in enterohemorrhagic Escherichia coli O157:H7[END_REF] . AIEC strains express LPF, which are known to target Peyer's patches in a mouse CD model [START_REF] Chassaing | Crohn disease-associated adherent-invasive E. coli bacteria target mouse and human Peyer's patches via long polar fimbriae[END_REF] . AIEC might therefore exploit EA as a metabolite but also coopt EA as a signaling molecule to recognize gastrointestinal environment and promote virulence expression. Further work is required to establish this. Nevertheless, the above-described accessory metabolic pathways may interact with synthetic pathways involved in biofilm formation. Indeed, these metabolic pathways can lead to the formation of acetate and pyruvate, which may participate in the formation of side chains of colanic acid. RNA-seq data analysis showed a global increase in the transcription of genes involved in the biofilm formation under bile conditions (genes involved in the formation of the colanic acid and of curli fimbriae, gene clusters encoding group 2 capsule [START_REF] Schembri | Capsule Shields the Function of Short Bacterial Adhesins[END_REF] , the auf and yeh genes involved in adhesion [START_REF] Antão | Adhesive threads of extraintestinal pathogenic Escherichia coli[END_REF] , …) (additional file 1: Table S2). In consistent with these data, the crystal violet assay revealed an increased of biofilm biomass and an autoaggregation of LF82 bacteria in presence of bile salts (additional file 1: Fig. S8). Thus, bile salts might favor AIEC gut colonization by inducing biofilm formation. Most strains of E. coli, Salmonella and Shigella forms a biofilm in the presence of bile salts; it is an important resistance mechanism for many bacterial pathogens [START_REF] Sistrunk | Survival of the Fittest: How Bacterial Pathogens Utilize Bile To Enhance Infection[END_REF][START_REF] Nickerson | Analysis of Shigella flexneri Resistance, Biofilm Formation, and Transcriptional Profile in Response to Bile Salts[END_REF] . Moreover, virulence factor expression of enteric pathogens and AIEC, is regulated during exposure to bile salts [START_REF] Chassaing | Bile salts induce long polar fimbriae expression favouring Crohn's disease-associated adherent-invasive Escherichia coli interaction with Peyer's patches[END_REF][START_REF] Gibold | The Vat-AIEC protease promotes crossing of the intestinal mucus layer by Crohn's disease-associated Escherichia coli[END_REF][START_REF] Sevrin | Adaptation of adherent-invasive E. coli to gut environment: Impact on flagellum expression and bacterial colonization ability[END_REF] . Given the dual control of resistance to bile salts and virulence factor expression in the presence of bile salts, it has been proposed that the enteric bile salt-induced biofilm is a transient phenotype that would allow a more rapid dispersion as the bacteria reach the epithelial cell surfaces in the small intestine [START_REF] Nickerson | Analysis of Shigella flexneri Resistance, Biofilm Formation, and Transcriptional Profile in Response to Bile Salts[END_REF] . The metabolic adaptation of AIEC presented in this work might be associated with this transient phenotype.

Studies examining the interactions between bacteria and bile have frequently been performed

with cholic acid and deoxycholic acid, which are among the most abundant in the intestine [START_REF] Begley | The interaction between bacteria and bile[END_REF] .

Since more than 20 bile acid metabolites can be produced from primary bile acids by the gut microbiota, the use of only two bide acids in our this study has limitation and needs further investigation. The study of Chassaing et al. [START_REF] Chassaing | Bile salts induce long polar fimbriae expression favouring Crohn's disease-associated adherent-invasive Escherichia coli interaction with Peyer's patches[END_REF] reported that the concentration of bile salts has no significant effect on lfp transcription, in contrast to the chemical nature of bile salts. That might be similar in our study. CD-associated intestinal dysbiosis leads to modifications in the luminal bile salt composition [START_REF] Duboc | Connecting dysbiosis, bile-acid dysmetabolism and gut inflammation in inflammatory bowel diseases[END_REF] . Thus, it is possible that the expression of AIEC genes (eut, pdu, lpf...) in the gut of CD patients is different from that of healthy subjects. In contrast to the study by Chassaing et al, we did not find a significant difference in LF82 lpf expression when LF82 was grown for 24 h at 37°C in a minimal medium (M9) with or without bile salts, suggesting that bacterial culture medium is also crucial for lpf gene expression. Further studies, which take these variables into account, are therefore necessary.

To conclude, in conjunction with previous work, our results support the notion that AIEC under bile conditions modulate expression of their virulence genes as well as secondary metabolic pathway genes to overcome nutritional limitations and compete with the indigenous microbiota.

Thus, our study highlight a metabolic adaptation of AIEC to bile salt exposure that may favor their colonization.

Methods

Bacterial strain and bacterial growth conditions

The bacterial strains used in this study are listed in additional file 1 (Methods, Table S3). The ability of E. coli LF82 to grow with bile salts and ethanolamine (EA) was tested on minimal medium mM9 as previously described [START_REF] Bertin | Enterohaemorrhagic Escherichia coli gains a competitive advantage by using ethanolamine as a nitrogen source in the bovine intestinal content[END_REF] (additional file 1: Methods). This medium was supplemented with bile salts (1%: 50% cholic acid sodium salt, 50% deoxycholic acid sodium salt, Sigma) when needed (mM9b).

For absolute quantification by RNA-seq, LF82 bacteria was grown for 24 h at 37°C in mM9 medium supplemented with D-glucose (0.1% w/v) and NH4Cl (20 mM) in a 20 mM Tris buffer with a pH = 7.5, in the presence or absence of 1% bile salts. Each assay was performed in triplicate. Utilization of EA as a nitrogen source or as the sole carbon source was investigated in the the mM9b medium supplemented with EA hydrochloride (mM9b-EA, additional file 1: Methods). For the other growth curves and for qRT-PCR of the eut genes, the growth medium used was the mM9-EA supplemented with 0.01% glucose. Bile salts (1%) were added or not in this medium. For quantification of metabolites in bacteria, E. coli strains were grown at 37°C to an OD600 of 0.7 in mM9 with 20% Lysogeny broth (LB). Then, bile salts (1%) were added or not for 4 h.

RNA extraction, reverse transcription and quantitative PCR

The AIEC reference strain LF82 was grown for 24 h at 37°C in a minimal medium with or without bile salts. Total RNAs were isolated from LF82 bacteria, and following rRNA depletion, the remaining RNA was reverse-transcribed into cDNA, fragmented and sequenced (for detailed Methods, see additional file 1: Methods). For qRT-PCR, extracted RNAs were quantified using a NanoPhotometer P-Class (Implen) and then reverse transcribed with random hexamers (Invitrogen) and NxGen M-MuLV Reverse Transcriptase (Lucigen) according to the manufacturer's recommendations. RNAs were then amplified using specific primers to the prpB, folX, pfkA, cfa, LF_715, eut, tufA or 16SrRNA genes (Aditional file 1: Methods, Table S1 and S4). Amplification of a single expected PCR product was confirmed by electrophoresis on a 2% agarose gel. qRT-PCR were performed using an Eppendorf Realplex with the following program: 95°C for 5 min and 40 cycles of 95°C for 15 s, 60°C for 20 s and 72°C for 40 s. For absolute qRT-PCR, the transcript levels in each sample were determined by absolute quantification using serial dilutions of PCR products as previously described [START_REF] Yoder-Himes | Mapping the Burkholderia cenocepacia niche response via highthroughput sequencing[END_REF] . Each experiment was performed in triplicate. For relative qRT-PCR, each gene expression was normalized to tufA using the 2 -ΔΔCt method.

RNA-seq data analysis

The RNA-seq data analysis was performed using the R software, Bioconductor packages including edgeR and the SARTools package developed at PF2-Institut Pasteur [START_REF] Varet | SARTools: A DESeq2-and EdgeR-Based R Pipeline for Comprehensive Differential Analysis of RNA-Seq Data[END_REF] (additional file 1: Methods).

Construction and transcomplementation of mutants

Isogenic mutants of E. coli LF82 was generated by using the lambda red recombination system described by Datsenko and Chaveroche [START_REF] Chaveroche | A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans[END_REF][START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF] (Additional file 1: Methods).

Competition experiments

Precultures of E. coli strains (inoculated from a single colony) were incubated in LB with the appropriate antibiotic (32 mg/L amoxicillin for LF82 bacteria and 50 mg/L kanamycin for LFΔeutB). The bacterial cultures were diluted 50-fold in LB broth and grown overnight at 37°C without shaking. The mM9b-EA medium previously used was supplemented with glucose (0.1%), inoculated with approximately 5.10 7 bacteria per milliliter of each of the two strains tested in competition assays and incubated at 37°C without shaking. At each time point, the coculture was diluted in phosphate-buffered saline (PBS) and spotted onto trypticase soy (TS) agar plates with and without antibiotic. Co-cultures of LF82/LFΔeutB bacteria were spotted on plates without antibiotic and on plates containing kanamycin (50 mg/L). Co-cultures of LF82 or LFΔeutB/MG1655 bacteria were spotted on plates without antibiotic and on plates containing amoxicillin (32 mg/L). The plates were then incubated overnight at 37°C before counting the colony-forming units (CFU). The CFU counts of the antibiotic-sensitive strain for each competition assay were calculated by subtracting the number of CFU that were resistant to amoxicillin or kanamycin from the number of CFU counted on an agar plate without antibiotic.

Each experiment was repeated three times, and a competitive index (CI) was calculated.

Metabolite extraction

Bacteria were harvested by centrifugation (10,000 g for ten minutes at 4°C). Quantified concentrations of metabolites were normalized to 3.10 10 bacteria. The supernatant was stored at -80°C, and the pellet was frozen at -20°C, thawed on ice, resuspended in cold extraction buffer (10 mM phosphate buffer, 10 mM MgCl2, 1 mM EDTA) and then disrupted by ultrasonic treatment (five times for 30 seconds). The extract was clarified by centrifugation at 10,000 g for 10 min. After addition of DNase I, the supernatant extract was centrifuged at 4°C, 10,000 g for 20 min, passed through a 0.2-µm filter and frozen to -80°C.

Metabolite quantification

LC/MS analyses were performed on a ThermoScientific UHPLC Ultimate 3000 RSLC coupled to an Orbitrap Q-Exactive analyser. The samples were diluted before the LC-MS analyses and injected directly in the LC-MS system without any further treatment. The calibration solutions were prepared from analytical standards dissolved in water. The UHPLC was equipped with a Luna Omega Polar C18 (100 × 2.1 mm; 1.6 µm) (Phenomenex) at 30°C with a gradient of acetonitrile + 0.1% formic acid and water + 0.1% formic acid. The analyses were carried out in negative mode (ESI -) with a spray voltage at 3.0 kV.

Murine model of gut colonization

The study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Université Clermont Auvergne. The animal protocol was approved by the committee for ethical issues, CEMEA Auvergne (Permit Number: CEMEAA, 2015032716314007), and all animals were used in accordance with the European Community Directive in the care and use of animals (86/609/CEE).

For in vivo competition assays, ten twelve-week-old C57BL/6 mice (body weight ≈26-28 g) [START_REF] Chan | Novel mouse model for carcinoembryonic antigen-based therapy[END_REF] were pretreated by administering oral antibiotics and 3% dextran sulfate sodium salt (Sigma) before being orally challenged with 10 9 bacteria (50% LF82 -50% LFΔeutB) (Supplementary information in addition file 1: Methods). Three days after bacterial infection, fresh fecal pellets (100-200 mg) were collected from individual mice and resuspended in PBS (addition file 1: Methods). Then, the mice were anesthetized with isoflurane and then euthanized by cervical dislocation. Colonization of the two strains was studied by enumerating the mucosa-associated AIEC bacteria by homogenizing 0.5 cm of ileum and 1 cm of colon, beginning at 0.5 cm from the cecal junction, in sterile PBS solution.

To determine the bacterial mRNA levels in the ileum of mice, the mice were pretreated by administering oral antibiotics for four days. From the day of infection until sacrifice, 2% cholestyramine was mixed with the drinking water, as recommended for human patients, in the group of treated mice. This suspension was regularly resuspended. All animals were orally challenged with 10 9 LF82 bacteria. The day after infection, the mice were anesthetized with isoflurane, euthanized by cervical dislocation and the ileum was removed.

Statistical analysis

Statistical comparisons were performed using the Mann-Whitney nonparametric test. An analysis of variance (ANOVA) was performed for experiments with multiple treatment groups, followed by pairwise comparisons with Bonferroni's multiple comparison tests. A p value less than 0.05 was considered statistically significant (* p<0.05; ** p<0.01; and *** p<0.001). For RNA-seq, mRNAs that were differentially expressed below the p-value threshold of 0.001 were considered significant. Gene expression from AIEC strain LF82 after growth in either mM9 minimal medium or mM9 supplemented with bile salts. The proportion of up-and downregulated genes (upon bile salts exposure relative to control) in major biological pathways of metabolism was compared to those in the whole transcriptome background using a hypergeometric test. A p value ≤0.05 was considered as significant. (A) Distribution of the upregulated and the downregulated genes in the different metabolic pathways. The percentage of LF82 genes involved in every metabolic pathway is indicated in pie chart. The percentage of increased transcript levels and of decreased transcript levels in every pathway are represented on the right: 62% genes encoding proteins involved in the degradation, utilization and assimilation of compounds were up-regulated (p<0,001) and 83% genes involved in biosynthesis pathways were down-regulated (p<0,001) in the presence of bile salts. (B) Distribution of the upregulated genes in main degradation pathways. The percentage of LF82 genes involved in each pathway is indicated in pie chart.

The percentage of increased transcript levels in every degradation pathways are presented on the right.

Metabolic adaptation of adherent-invasive Escherichia coli to exposure to bile salts

Julien Delmas* 1,2 , Lucie Gibold* 1,2 , Tiphanie Faïs France (approval number AU 904). Suspicious E. coli colonies were identified by MALDI-TOF mass spectrometry (bioMérieux, France). Determination of E. coli strains as belonging to the AIEC pathovar was performed using the following criteria: (1) the ability to adhere to I-407 epithelial cells with an adhesion index equal or superior to 1 bacteria per cell, (2) the ability of the bacteria to invade I-407 with an invasion index equal or superior to 0.1% of the original inoculum [START_REF] Martin | Enhanced Escherichia coli adherence and invasion in Crohn's disease and colon cancer[END_REF] .

Strain LF82, isolated from a patient with ileal CD, is the archetypal AIEC strain, belongs to the E. coli serotype O83:H1 2 (additional file 1: Table S3). This strain contains cephalosporinase and can be selected on amoxicillin-containing agar plates. The non-pathogenic E. coli K-12 strain MG1655 does not display the AIEC phenotype.

Bacterial growth conditions

A modified M9 minimal medium (mM9) containing Na2HPO4 (48 mM), KH2PO4 (22 mM),

and NaCl (8.5 mM) was supplemented with MgSO4 (1 mM), CaCl2 (0.1 mM), vitamin B12

(cyanocobalamin) (150 nM), vitamin B1 (thiamine, 5 mg/L) and trace metals (0.1 µM ZnSO4, 0.045 µM FeSO4, 0.2 µM Na2Se2O3, 0.2 µM Na2MoO4, 2 µM MnSO4, 0.1 µM CuSO4, 3 µM CoCl2 and 0.1 µM NiSO4) (2 mL/L). This medium was supplemented with bile salts (1%: 50% cholic acid sodium salt, 50% deoxycholic acid sodium salt, Sigma) when needed (mM9b).

Utilization of EA as a nitrogen source was investigated by addition of EA hydrochloride (5 mM) and glucose (0.1 %) to the mM9b medium (mM9b-EA). To test the capacity of the LF82 strain to use EA as the sole carbon source, the mM9b medium was supplemented with EA hydrochloride (5 mM) and NH4Cl (20 mM). For each condition, three lysogeny broth (LB) cultures were each started from a single colony and grown overnight at 37°C with aeration.

Cells were pelleted by centrifugation, resuspended in medium with EA as the sole nitrogen or carbon source and diluted 50-fold in the corresponding medium. Cultures were then incubated at 37°C, and growth was monitored in three parallel cultures by following the optical density (λ=600 nm).

RNA extraction

Total RNAs were extracted from bacteria using a Direct-zol RNA MiniPrep kit (Zymo research)

and treated with a Turbo DNA-free kit (Ambion) to remove any contaminating genomic DNA.

DNase-treated RNA samples were purified with RNA clean and concentrator-25 (Zymo research). For RNA-seq analysis, purified RNAs were then quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific). The integrity of the results (RNA integrity number

[RIN]) was assessed using an Agilent 2100 bioanalyzer. A Ribo-zero Magnetic kit for Gramnegative bacteria (Epicentre) was used according to the manufacturer's recommendations to remove the 23S and 16S rRNA from the total RNA samples. The samples were then purified using RNA clean and concentrator-5 (Zymoresearch). To evaluate the degree of rRNA depletion, the samples were analyzed using an Agilent 2100 bioanalyzer. The remaining RNA was sequenced using Illumina HiSeq 2500 technology with the genomic ProfileXpert platform (Claude Bernard University, Lyon, France). Three replicates from each experimental condition (with or without bile salts) were employed.

RNA-seq data analysis

We used RNA-seq to compare transcriptomes to understand the regulatory networks that control gene expression in AIEC strains during ileal colonization. Briefly, reads were mapped against the genomic sequence of E. coli LF82 (GenBank accession n° NC_011993; 3 ). Reads mapped to several positions and reads mapped to rRNA were removed from further analysis.

The number of reads overlapping each gene based on GenBank annotation was recorded. Reads from replicate samples were pooled, and the number of reads per gene was normalized according to the total number of reads in each library and the gene size. To normalize the expression of genes in different RNA-seq samples, values corresponding to the number of reads per kilobase per million mapped (RPKM) were calculated as follows: (number of reads for the gene x 10 9 ) / (total number of reads x size of the gene). A p-value adjustment for differentially expressed genes (DEGs) was performed to take into account multiple testing and control the false positive rate to a chosen level α < 0.001. Products of DEGs were classified by functional category according to metabolism pathways of the Ecocyc database (http://ecocyc.org) [START_REF] Baumgart | Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum[END_REF] . The RKPM values for each gene were plotted and visualized as a circle using the Circos program 5 .

Metabolic pathways were analyzed using Ecocyc [START_REF] Darfeuille-Michaud | Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease[END_REF] .

A total of 53,900,605 reads from minimal medium and 56,327,631 reads from medium supplemented with bile salts were obtained for each cDNA library. Among them, 50,353,549 (93.4%) and 53,246,854 (94.5%) reads mapped to the genome of Escherichia coli LF82, which has a total size of 4,773,108 bp. The average numbers of reads per region were 462 (without bile salts) and 747 (with bile salts), with a coverage of 98.4% and 98.6% of the 4376 encoding DNA-encoding sequences (CDS) of E. coli LF82 represented by at least one single read. The absolute and relative distributions of reads in the two media for the annotated genes of the LF82 strain are shown in Fig. S1. A dendrogram used to represent all the samples shows the grouping of the replicates and a difference between the biological conditions. Box plots and the volcano plot are also represented to show the quality of the normalization and the differential expression of genes (Fig. S1). To assess the reliability of RNA-seq for determining the relative abundances of individual transcripts in the absence and presence of bile salts, we used absolute quantification of mRNAs for three up-regulated genes (prpB, eutB and LF82_715), two downregulated genes (pfkA and cfa) and one unchanged gene (folX) by qRT-PCR. The data for these genes provided an r2 value of 0.9708, confirming the data obtained by RNA-seq (Fig. S2; Table S1).

Construction and transcomplementation of isogenic mutants

Isogenic mutants of E. coli LF82 was generated by using the lambda red recombination system.

E. coli LF82 was transformed with pKOBEG, a plasmid encoding the Red proteins that protect linear DNA from degradation in bacteria. The plasmid was maintained in bacteria at 30°C with 25 mg/l of chloramphenicol and 1 mM of L-arabinose. The Flp recognition target-flanked cassette harboring the kanamycin resistance cassette was generated by PCR from E. coli S2)

BW25141 with d-eutB-F/d-eutB-R, d-eutE-F/d-eutE-R and d-citF-F/citF-R primers (Table
and High Fidelity Platinum Taq polymerase (Invitrogen) according to the manufacturer's instructions. The PCR products were electroporated into previously glycerol-washed E. coli LF82. The resulting LF82ΔeutB, LF82ΔeutE and LF82 ΔcitF isogenic mutants (Km R ) were selected on LB agar containing 50 mg/L kanamycin. Replacement of the eut and citF genes by the kanamycin resistance cassette was confirmed by PCR. The kanamycin resistance cassette was then removed from LF82ΔeutB bacteria by the transient expression of the Flp recombinase from the pCP20 plasmid, creating the LF82ΔeutB (Km S ) strain.

The eutB gene was amplified by PCR from E. coli LF82 genomic DNA using eutBEcoR1-F and eutBBamH1-R primers (Table S2). The amplified DNA was purified with a NucleoSpin extract kit (Macherey-Nagel), digested with EcoRI and BamHI (New England Biolabs), and ligated to the EcoRI-BamHI-digested expression vector pBK-CMV (Agilent Technologies).

This construct was electroporated into LF82ΔeutB (Km S ) electrocompetent strains and selected on Mueller Hinton agar containing 50 mg/L kanamycin. The presence of the eutB gene was confirmed by PCR. The construction was checked by double-stranded DNA sequencing (GATC biotech, Germany).

Murine model of gut colonization

For the in vivo experiments, we used C57BL/6 mice, which were housed in specific pathogenfree conditions in the animal care facility at the Université Clermont Auvergne, Clermont-Ferrand, France. For in vivo competition assays, ten twelve-week-old mice (body weight ≈26-28 g) were pretreated by administering oral amoxicillin (1 g/L), vancomycin (500 mg/L), metronidazole (500 mg/L) and neomycin (1 g/L) for four days, and 3% dextran sulfate sodium salt (Sigma) for the last day. At 24 h after stopping the antibiotic treatment, the animals were orally challenged with 10 9 bacteria (50% LF82 -50% LFΔeutB). Three days after bacterial infection, fresh fecal pellets (100-200 mg) were collected from individual mice and resuspended in PBS. After serial dilutions, the bacteria were enumerated by plating on TS agar medium containing amoxicillin to isolate the two bacteria and amoxicillin + kanamycin to isolate LFΔeutB. The plates were then incubated overnight at 37°C before counting the CFU.

The CFU count of the LF82 strain for each mouse was calculated by subtracting the number of CFU that were resistant to kanamycin from the number of CFU counted on an agar plate containing only amoxicillin. Three days after infection, the mice were anesthetized with isoflurane and then euthanized by cervical dislocation. Colonization of the two strains was studied by enumerating the mucosa-associated AIEC bacteria by homogenizing 0.5 cm of ileum and 1 cm of colon, beginning at 0.5 cm from the cecal junction, in sterile PBS solution. Samples were plated on TS agar containing amoxicillin or amoxicillin + kanamycin and incubated overnight at 37°C.

Biofilm formation assay

Biofilm assays were used as previously described with some modifications [START_REF] Glasser | Adherent Invasive Escherichia coli Strains from Patients with Crohn's Disease Survive and Replicate within Macrophages without Inducing Host Cell Death[END_REF][START_REF] Eaves-Pyles | Escherichia coli isolated from a Crohn's disease patient adheres, invades, and induces inflammatory responses in polarized intestinal epithelial cells[END_REF] . Briefly, 6 µL of a 3-hour culture in mM9-EA medium supplemented or without 1% bile salts was inoculated into 144 µL of the same medium in a 96-well culture-treated polystyrene microtiter plate (Nunc). Wells filled with growth medium alone were included as negative controls. After 4 h and 30 min of incubation at 37°C, surface-adherent biofilm formation was measured by staining bound cells for 15 min with a 0.5% (w/v) aqueous solution of crystal violet. After rinsing with distilled water, the bound dye was released from the stained cells using 95% ethanol, and the OD at 540 nm was determined.

Autoaggregation assay

After an overnight culture at 37°C in M9 minimal medium supplemented with glucose (0.1%) and bile salts (1%) when needed, cells were diluted in the same medium and grown at 37°C to the exponential phase. Next, 20 µL of each culture was Gram-stained and visualized by microscopy. Ethanolamine is metabolized by AIEC to produce nitrogen and acetyl-CoA. Citrate is converted to acetate and oxaloacetate by citrate lyase. Oxaloacetate, which is necessary for the methylcitrate pathway, is also supplied for L-tartrate fermentation and amino acid degradation.

Supplemental data

Propionyl-coA from various degradation products is metabolized to produce pyruvate, which can be converted to acetyl-coA. The genes that exhibited an increase in mRNA concentrations in bacteria grown with bile salts in comparison to without bile salts encoded proteins involved in ethanolamine degradation (green boxes), 2-methylcitrate pathway (blueberry boxes), citrate and L-tartrate fermentation (cyan boxes) and 1,2-propandediol degradation (pink boxes).

Figure 1 :

 1 Figure 1: Bile salts induce a global up-regulation of LF82 genes involved in degradation

Figure 2 :

 2 Figure 2: AIEC strain LF82 can use ethanolamine as a sole nitrogen source in presence of

Figure 3 :

 3 Figure 3: Comparison of ethanolamine utilization in AIEC and non-AIEC strains after

Figure 4 :

 4 Figure 4: Ethanolamine utilization by AIEC strain LF82 provides a growth advantage

Figure 5 :

 5 Figure 5: eutB favors AIEC strain LF82 gut colonization in mice.

Figure 6 :

 6 Figure 6: Bile salts increase the intracellular acetyl-CoA pool in the LF82 strain.

Figure 7 :

 7 Figure 7: Alternative metabolic pathways in AIEC strain LF82 are induced in the small

Figure S1 :

 S1 Figure S1: Global data retrieved by RNA-seq. (A) Dendrogram built based on the Ward criteria, which show variability in raw data within the experimental conditions. (B) Boxplots of raw (left) and normalized (right) data to assess the quality of the normalization process. (C) Volcano plot (bile salts containing medium versus medium) representing the log of the adjusted p value as a function of the log ratio of differential expression.

Figure S2 :

 S2 Figure S2: Analysis and validation of the RNA-seq experiments.(A) Global analysis of transcript levels in E. coli LF82 by RNA-seq. The gray circular lines represent a log2 = 1 (i.e., RPKM bile salts containing medium/minimal medium ratio values) of each gene among the upregulated genes (yellow background) and downregulated genes (green background). Black and blue circles on a white background correspond to the expression of each gene (represented as RPKM values) in bacteria grown under minimal conditions and bile salts-containing medium, respectively. The outermost circle represents the 4,773,108 bp of the LF82 genome. (B) Validation of the RNA-seq results by qRT-PCR for selected genes. Mean log2 ratios of values determined in the qRT-PCR experiments are plotted against the mean log2 ratios of values determined in the RNA-seq experiments (Supporting Information TableS1). (C) Global analysis of increased transcript levels of genes involved in metabolism in E. coli LF82 with connections with each metabolic pathway. The gray circular lines represent a log2 = 0.5 (i.e., RPKM bile salts containing medium/minimal medium ratio values) of each gene among the upregulated genes. (D) Global analysis of decreased transcript levels of genes involved in metabolism in E. coli LF82 with associations with each metabolic pathway. The gray circular lines represent a log2 = 0.5 (i.e., RPKM bile salts containing medium/minimal medium ratio values) of each gene among the downregulated genes.

Figure S3 :

 S3 Figure S3: Schematic pathways of ethanolamine catabolism (A), citrate degradation (B)

Figure S4 :

 S4 Figure S4: Comparison of growth of AIEC and non-AIEC strains in minimal medium.

Figure S5 :

 S5 Figure S5: Bacterial growth curves of LF82 and MG1655 strains in LB and mM9-EA supplemented with bile salts. Values are means ± SEM of at least three independent experiments. The growth difference between both media was 68 and 90% for LF82 and MG1655, respectively.

Figure S6 :

 S6 Figure S6: Schematic showing the interplay between ethanolamine degradation, citrate

Figure S7 :

 S7 Figure S7: Effect of cholestyramine on mRNA levels of shp and ibabp in the ileum of mice. Cholestyramine is a bile-acid sequestrant that binds bile acids in the intestine to prevent their reabsorption. As a result, this decreases the feedback suppression of hepatic bile-acid synthesis from cholesterol. Values are means ± SEM. Statistical analysis was performed with a Mann-Whitney test; * p<0.05.

Figure S8 :

 S8 Figure S8: Bile salts induce biofilm formation and autoaggregation of bacteria.

Table 1 : Variations of the expression of eut genes

 1

			RNA-seq values	RNA-seq values
	name	gene size	(minimal medium)	(bile salts)	Fold change Fold change	p value
		(bp)	RPKM	RPKM		(Log2)
	eutA	1403	36	223	6.3	2.65	2.151E-13
	eutB	1361	61	358	5.9	2.55	1.818E-17
	eutC	887	47	286	6.1	2.60	5.747E-17
	eutD	1016	108	357	3.3	1.72	2.746E-11
	eutE	1403	14	149	10.4	3.38	1.849E-16
	eutG	1187	35	168	4.9	2.28	2.691E-11
	eutH	1226	106	514	4.8	2.27	1.172E-15
	eutJ	836	13	147	11.3	3.50	8.290E-18
	eutK	500	37	94	2.5	1.34	9.218E-05
	eutL	659	30	126	4.2	2.08	1.825E-09
	eutM	335	55	177	3.2	1.69	1.128E-05
	eutN	287	5	62	12.2	3.61	1.249E-12
	eutP	479	39	166	4.2	2.08	7.469E-10
	eutQ	701	24	192	8.1	3.01	1.052E-12
	eutR	1052	142	308	2.2	1.12	3.639E-05
	eutS	335	20	76	3.9	1.96	8.018E-07
	eutT	803	13	83	6.4	2.68	6.423E-10
	1 values shown in bold are significant

Table 2 : Variations of the expression of genes involved in glycolysis, TCA cycle and formation of acetyl-coA

 2

			gene	RNA-seq values	RNA-seq values	Fold	Fold
	name	product	size	(minimal medium)	(bile salts)	change	change	p value
			(bp)	RPKM	RPKM		(Log2)
	aceE	Pyruvate dehydrogenase E1	2663	6420	11797	-1.8	-0.88	1.188E-02
	aceF	Dihydrolipoyllysine-residue acetyltransferase	1892	2620	3886	-1.5	-0.57	6.618E-02
	ackA	acetate kinase	1202	2740	4056	-1.5	-0.57	7.020E-02
	acs	acetyl-coenzyme A synthetase	1958	2188	837	2.6	1.39	1.897E-04
	eno	Enolase	1298	9777	15019	-1.5	-0.62	6.921E-02
	fbaA	Fructose-bisphosphate aldolase class 2 1079	3314	3828	-1.2	-0.21	4.847E-01
	fbaB	Fructose-bisphosphate aldolase class 1 1124	943	837	1.1	0.17	4.857E-01
	fbp	Fructose-1.6-bisphosphatase	998	3314	5750	-1.7	-0.79	1.384E-02
	fumA	Fumarate hydratase class I. aerobic	1646	7038	9348	-1.3	-0.41	1.869E-01
	fumB	Fumarate hydratase class I. anaerobic	1646	771	415	1.9	0.90	5.607E-05
	fumC	Fumarate hydratase class II	1403	1122	884	1.3	0.34	1.468E-01
	gapA	glyceraldehyde-3-phosphate dehydrogenase A	995	25736	36899	-1.4	-0.52	1.405E-01
	glk	glucokinase	965	832	912	-1.1	-0.13	6.037E-01
	gltA	Citrate synthase	1283	7479	15175	-2.0	-1.02	7.423E-04
		bisphosphoglycerate-dependent
	gpmA	phosphoglycerate	752	10216	19188	-1.9	-0.91	4.772E-03
		bisphosphoglycerate-independent
	gpmM	phosphoglycerate	1544	1282	1810	-1.4	-0.50	7.967E-02
	icd	Isocitrate dehydrogenase [NadP]	1250	17987	30473	-1.7	-0.76	1.944E-02
	lpd	Dihydrolipoyl dehydrogenase	1424	10039	20656	-2.1	-1.04	1.135E-03
	mdh	Malate dehydrogenase	938	5402	8506	-1.6	-0.66	2.854E-02
	pfkA	6-phosphofructokinase isozyme 1	962	1283	4244	-3.3	-1.72	1.369E-11
	pgi	glucose-6-phosphate isomerase	1649	4480	5251	-1.2	-0.23	4.652E-01
	pgk	Phosphoglycerate kinase	1163	2762	4408	-1.6	-0.67	3.299E-02
	pta	Phosphate acetyltransferase	2144	1355	1764	-1.3	-0.38	1.523E-01
	pykA	Pyruvate kinase II	1442	1163	1360	-1.2	-0.23	3.798E-01
	pykF	Pyruvate kinase I	1412	2019	5004	-2.5	-1.31	7.772E-07
		Succinate dehydrogenase flavoprotein
	sdhA	subunit	1766	8036	14377	-1.8	-0.84	1.123E-02
		Succinate dehydrogenase iron-sulfur
	sdhB	subunit	716	4221	6984	-1.7	-0.73	2.521E-02
		Succinate dehydrogenase cytochrome
	sdhC	b556 subunit	389	4494	8916	-2.0	-0.99	1.946E-03
		Succinate dehydrogenase hydrophobic
	sdhD	membrane	347	2126	3986	-1.9	-0.91	6.527E-04
		2-oxoglutarate dehydrogenase E1
	sucA	component	2801	18453	38038	-2.1	-1.04	1.922E-03
		Dihydrolipoyllysine-residue
	sucB	succinyltransferase	1217	9625	19122	-2.0	-0.99	5.780E-03
	sucC	Succinyl-CoA synthetase beta chain	1168	7109	12688	-1.8	-0.84	1.840E-02
		Succinyl-CoA ligase [adP-forming]
	sucD	subunit α	869	9683	12034	-1.2	-0.31	3.924E-01
	tpiA	Triosephosphate isomerase	767	4649	4399	1.1	0.08	8.098E-01
		1 values shown in bold are significant

Table 3 : Variations of the expression of genes involved in citrate degradation and methyl- citrate pathway

 3

					RNA-seq
				RNA-seq values	values (bile	Fold	Fold
	name	product	gene size	(minimal medium)	salts)	change	change	p value
			(bp)	RPKM	RPKM		(Log2)
	acnA aconitate hydratase 1	2675	1824	2317	-1.3	-0.34	2.142E-01
	acnB aconitate hydratase 2	2597	11663	19016	-1.6	-0.70	2.362E-02
	acs acetyl-coenzyme A synthetase	1958	2188	837	2.6	1.39	1.897E-04
	citC Citrate-lyase ligase	1058	326	588	1.8	0.85	8.689E-04
	citD Citrate lyase acyl carrier protein	296	5	50	9.1	3.18	3.731E-10
	citE Citrate lyase  subunit	908	48	332	7.0	2.80	1.628E-17
	citF Citrate lyase α chain	1532	55	362	6.6	2.72	8.791E-16
	citG dephosphocoenzyme-A	878	32	183	5.6	2.49	5.951E-14
	citT Citrate carrier	1463	271	732	2.7	1.43	2.076E-09
	citX Holo-citrate lyase synthase	551	5	59	10.6	3.41	1.042E-13
	ttdA L-tartrate dehydratase subunit α	908	237	49	4.8	2.27	2.511E-13
	ttdB L-tartrate dehydratase subunit 	605	317	66	4.8	2.27	6.822E-12
	ttdR transcriptional activator	932	635	341	1.9	0.90	5.398E-04
	ttdT tartrate carrier	1463	724	312	2.3	1.21	1.002E-06
	prpR operon regulatory protein	1586	192	435	2.3	1.19	1.071E-04
	prpB Methylisocitrate lyase	890	131	3094	23.6	4.56	1.828E-37
	prpC 2-methylcitrate synthase	1169	106	1313	12.3	3.62	3.179E-30
	prpD 2-methylcitrate dehydratase	1451	66	609	9.3	3.21	2.654E-26
	prpE Propionate-CoA ligase	1886	80	786	9.8	3.30	5.999E-33

Table 4 : Variations of the expression of genes involved in rhamnose and 1,2-propanediol degradation

 4

			gene	RNA-seq values	RNA-seq values	Fold	Fold
	name	product	size	(minimal medium)	(bile salts)	change	change	p value
			(bp)	RPKM	RPKM		(Log2)
	rhaA	L-rhamnose isomerase	1259	376	39	9.7	3.27	2.279E-24
	rhaB	Rhamnulokinase	1469	452	65	7.0	2.80	3.564E-19
	rhaD	Rhamnulose-1-phosphate aldolase	824	554	303	1.8	0.87	3.341E-03
	fucO	propanediol oxydoreductase	1151	510	234	2.2	1.12	5.724E-06
	pduB	polyhedral bodies	812	71	203	2.8	1.51	3.202E-07
	pduC	glycerol dehydratase large subunit 1664	251	532	2.1	1.08	3.910E-06
	pduD	diol dehydratase medium subunit	668	110	236	2.2	1.11	5.478E-04
	pduE	diol dehydratase small subunit	518	39	118	3.1	1.62	7.898E-06
	pduG	diol dehydratase reactivation	1832	185	494	2.7	1.42	8.661E-08
	pduH	diol dehydratase reactivation	350	71	174	2.4	1.29	4.274E-05
	pduJ	polyhedral bodies	275	42	135	3.2	1.69	2.362E-07
	pduK	polyhedral bodies	422	16	59	3.7	1.89	1.644E-06
	pduL	Phosphate propanoyltransferase	632	53	134	2.6	1.35	5.949E-05
	pduM	propanediol utilization protein	491	78	233	3.0	1.59	2.260E-07
	pduN	polyhedral bodies	275	48	90	1.9	0.91	1.456E-02
	pduO propanediol utilization: B12 related 1007	106	261	2.5	1.30	6.990E-06
	pduP	propionaldehyde dehydrogenase	1112	81	211	2.6	1.39	2.872E-06
	pduQ	Propanol dehydrogenase	1343	125	416	3.3	1.74	3.516E-09
	pduT	polyhedral bodies	554	55	157	2.8	1.51	1.503E-05
	pduU	polyhedral bodies	350	95	102	1.1	0.11	7.703E-01
	pduV	propanediol utilization protein	443	188	152	-1.2	-0.31	2.703E-01

  [START_REF] Martin | Enhanced Escherichia coli adherence and invasion in Crohn's disease and colon cancer[END_REF][START_REF] Darfeuille-Michaud | High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease[END_REF] , Sylvine Batista 1 , Martin Leremboure 4 , Clara Sinel 3 , Emilie Vazeille 2 , Vincent Cattoir 3 , Anthony Buisson 2,5 , NicolasBarnich 2,[START_REF] Darfeuille-Michaud | Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease[END_REF]

	Guillaume Dalmasso 2 , Richard Bonnet 1,2
	Supplemental Methods
	Bacterial strains

E. coli were isolated from ileal biopsies of patients. In this prospective multicentre study (8 centers), all the patients requiring ileocolonoscopy, regardless of the indication, were consecutively included between September 2015 and September 2016. The study was performed in accordance with the Declaration of Helsinki, Good Clinical Practice and applicable regulatory requirements. Study ethics approval was obtained on CPP Sud-Est 6,

Table S1 : qRT-PCR validation of selected genes

 S1

				qRT-PCR	RNA-seq
	Gene
	number	Gene name Product name	fold change	fold change
				(Log2)	(Log2)
	LF82_0581	eutB	ethanolamine ammonia-lyase heavy chain	1.882	2.55
	LF82_1739	prpB	Methylisocitrate lyase	2.59	4.561
			D-erythro-7.8-dihydroneopterin triphosphate
	LF82_0729	folX		-0.802	-0.262
			epimerase
	LF82_1619	pfkA	6-phosphofructokinase isozyme 1	-2.43	-1.725
	LF82_0286	cfa	Cyclopropane-fatty-acyl-phospholipid synthase	-2.70	-2.70
	LF82_p715	LF82_p715 Ornithine carbamoyltransferase chain I	1.383	1.99

Table S2 : Variations of the expression of genes involved in biofilm formation

 S2

				RNA-seq values RNA-seq values RNA-seq values	RNA-seq values (bile RNA-seq values (bile RNA-seq values (bile	Fold Fold Fold	Fold Fold Fold
	name name name	product product product	gene size gene size gene size	(minimal medium) (minimal medium) (minimal medium)	salts) salts) salts)	change change change	change change change	p value p value p value	type of virulence gene type of virulence gene type of virulence gene
			(bp) (bp) (bp)	RNA-seq values RPKM RPKM RPKM	RNA-seq values (bile RPKM RPKM RPKM	Fold	Fold (Log2) (Log2) (Log2)
	name gmd ydeR LF82_p470	product GDP-mannose 4.6-dehydratase fimbrial-like protein ydeR orf	gene size 1121 503 1190	(minimal medium) 321 339 430	salts) 67 421 709	change 4.8 1.2 1.6	change 2.256 0.315 0.72	p value 7.842E-12 2.302E-01 6.277E-04	type of virulence gene biofilm pili PAI-IV/group 2 capsul
	ppdD ydes LF82_p471	Prepilin peptidase-dependent protein D fimbrial-like protein ydes orf	(bp) 440 530 1250	RPKM 116 181 212	RPKM 128 323 476	1.1 1.8 2.3	(Log2) 0.14 0.83 1.17	6.745E-01 7.688E-03 8.775E-04	pili pili PAI-IV/group 2 capsul
	acrE tnaA yehA LF82_p472	acriflavine resistance protein E Tryptophanase orf glycerol-3-phosphate cytidyltransferase	1157 1430 1034 395	619 2970 351 137	127 545 465 490	4.9 5.5 1.3 3.6	2.29 2.45 0.41 1.842	1.181E-15 8.555E-15 1.382E-01 5.837E-14	biofilm other pili PAI-IV/group 2 capsul
	acrF tnaB yehB LF82_p473	acriflavine resistance protein F Low affinity tryptophan permease outer membrane usher protein yehB KpsT protein	3104 1247 2480 674	1355 1573 243 119	665 558 822 280	2.0 2.8 3.4 2.4	1.03 1.495 1.761 1.233	2.395E-05 4.406E-11 2.152E-11 1.382E-05	biofilm other pili PAI-IV/group 2 capsul
	bcsA bcsB wcaA yehC LF82_p474	Cellulose synthase catalytic subunit [UDP-forming] Cyclic di-GMP-binding protein colanic acid biosynthesis glycosyl transferase fimbrial chaperone yehC KpsM protein	2618 2339 839 674 776	2103 993 264 27 250	1498 612 52 223 498	1.4 1.6 5.1 8.1 2.0	0.49 0.70 2.34 3.02 1.00	5.361E-02 2.248E-03 1.053E-11 7.172E-18 6.626E-05	biofilm biofilm biofilm pili PAI-IV/group 2 capsul
	bcsC wcaB yehD LF82_p509	Cellulose synthase operon protein C colanic acid biosynthesis acetyltransferase orf Yqi fimbriae subunit YgiL	3473 488 542 563	1450 109 28 129	978 22 124 206	1.5 4.9 4.4 1.6	0.57 2.305 2.14 0.69	1.407E-02 5.687E-10 5.677E-11 1.835E-02	biofilm biofilm pili pili
	bcsZ wcac yehE LF82_p510	endoglucanase colanic acid biosynthesis glycosyl transferase orf outer membrane usher protein YqiG	1112 1217 281 2522	693 405 438 1748	519 63 452 1134	1.3 6.4 1.0 -1.5	0.42 2.684 0.04 -0.62	6.640E-02 2.750E-16 8.737E-01 8.810E-03	biofilm biofilm pili pili
	cpsB wcad LF82_p366 LF82_p511	Mannose-1-phosphate guanylyltransferase [GDP] colanic acid polymerase orf fimbrial chaperone yqiH	1436 1217 857 749	448 656 683 779	83 150 608 620	5.4 4.4 -1.1 -1.3	2.44 2.13 -0.169 -0.33	4.227E-15 6.018E-13 4.454E-01 1.175E-01	biofilm biofilm pili pili
	cpsG wcae LF82_p367 LF82_p512	Phosphomannomutase (colanic acid biosynthesis) colanic acid biosynthesis glycosyl transferase fimbrial-like protein yfcP Yqi fimbrial adhesin	1370 746 446 1049	160 275 40 3757	25 60 149 1483	6.3 4.6 3.7 -2.5	2.65 2.202 1.89 -1.34	2.972E-14 5.112E-12 1.704E-08 1.502E-06	biofilm biofilm pili pili
	csgA wcaF LF82_p368 LF82_p527	Major curlin subunit colanic acid biosynthesis acetyltransferase Yfc fimbriae subunit YfcQ fimbrial adhesin (auf)	455 548 479 1130	74 116 39 190	206 26 99 558	2.8 4.4 2.5 2.9	1.48 2.14 1.33 1.552	4.550E-06 8.126E-09 1.296E-04 4.169E-09	pili biofilm pili pili
	csgB wcaI LF82_p369 LF82_p528	Minor curlin subunit colanic acid biosynthesis glycosyl transferase orf fimbrial chaperone (auf)	455 1214 503 749	67 241 28 116	138 25 47 377	2.1 9.8 1.7 3.3	1.05 3.29 0.74 1.71	8.281E-04 7.506E-18 8.409E-02 1.416E-09	pili biofilm pili pili
	fcl wcaJ LF82_p370 LF82_p529	GDP-L-fucose synthetase colanic biosynthesis UDP-glucose lipid carrier periplasmic chaperone YfcS minor fimbrial subunit precursor (auf)	965 1394 752 515	163 461 100 129	41 65 261 277	4.0 7.0 2.6 2.1	1.99 2.81 1.38 1.103	6.025E-09 1.056E-17 5.212E-06 1.024E-04	biofilm biofilm pili pili
	fimA wcaK LF82_p371 LF82_p530	Type-1 fimbrial protein. A chain colanic acid biosynthesis protein wcaK outer membrane usher YfcU minor fimbrial subunit precursor (auf)	548 1280 2654 563	7094 244 980 187	21306 58 1178 481	3.0 4.2 1.2 2.6	1.59 2.08 0.27 1.361	1.812E-06 3.060E-10 3.152E-01 4.641E-07	pili biofilm pili pili
	fimB wcal LF82_p372 LF82_p531	Type 1 fimbriae regulatory protein fimB colanic acid biosynthesis glycosyltransferase Yfc fimbriae subunit YfcV Outer membrane usher protein AufC	602 1220 566 2594	413 449 429 422	428 564 143 1586	1.0 -1.3 -3.0 3.8	0.05 -0.33 -1.58 1.91	8.898E-01 1.687E-01 2.635E-08 1.131E-11	pili biofilm pili pili
	fimC wcaM LF82_p461 LF82_p532	chaperone protein fimC colanic acid biosynthesis protein wcaM orf Auf fimbiral chaperone 1	725 1394 983 752	2840 826 140 324	3333 546 680 487	1.2 1.5 4.8 1.5	0.23 0.598 2.273 0.589	4.971E-01 1.219E-02 8.694E-21 1.275E-02	pili biofilm PAI-IV/group 2 capsul pili
	fimD wza LF82_p462 LF82_p533	Outer membrane usher protein fimD polysaccharide export protein wza Polysialic acid capsule synthesis protein KpsE	2636 1139 1148 704	3624 349 187 882	6328 74 629 621	1.7 4.7 3.4 -1.4	0.80 2.226 1.75 -0.51	9.777E-03 5.062E-13 1.511E-15 2.276E-02	pili biofilm PAI-IV/group 2 capsul pili
	fimE wzb LF82_p463 LF82_p548	Type 1 fimbriae regulatory protein fimE Low molecular weight protein-tyrosine-phosphatase KpsD protein major fimbrial subunit	596 443 1676 524	809 107 260 180	450 20 802 294	-1.8 5.4 3.1 1.6	-0.84 2.434 1.625 0.71	1.584E-04 9.992E-08 2.746E-14 1.020E-02	pili biofilm PAI-IV/group 2 capsul pili
	fimF wzc LF82_p464 LF82_p549	Protein fimF Tyrosine-protein kinase wzc 3-deoxy-manno-octulosonatecytidylyltransferase fimbriae	530 2162 740 1052	206 650 54 1495	415 139 296 1122	2.0 4.7 5.4 -1.3	1.008 2.23 2.443 -0.414	1.064E-04 9.343E-17 1.120E-17 1.111E-01	pili biofilm PAI-IV/group 2 capsul pili
	fimG yadC LF82_p465 LF82_p550	Protein fimG fimbrial-like protein yadC KpsC protein Outer membrane usher protein lpfC precursor	503 1262 2027 2531	208 1607 324 1297	528 1611 1002 1444	2.5 1.0 3.1 1.1	1.34 0.004 1.627 0.154	4.303E-08 9.912E-01 4.229E-14 5.427E-01	pili pili PAI-IV/group 2 capsul pili
	fimH yadK LF82_p466 LF82_p551	Protein fimH orf KpsS protein Fimbrial chaperone protein	902 596 1259 686	574 103 246 96	920 271 916 193	1.6 2.6 3.7 2.0	0.681 1.392 1.90 1.00	5.124E-03 1.260E-01 4.083E-18 1.393E-03	pili pili PAI-IV/group 2 capsul pili
	fimI yadL LF82_p467 LF82_p552	Fimbrin-like protein fimI orf Transposase within prophage fimbrial-like protein	539 608 1124 524	3699 92 212 244	5088 160 547 188	1.4 1.8 2.6 -1.3	0.46 0.81 1.37 -0.37	2.335E-01 3.809E-03 2.025E-08 1.943E-01	pili pili PAI-IV/group 2 capsul pili
	flhC ydeP LF82_p468	flagellar transcriptional activator flhC Protein ydeP orf	578 2279 983	1026 568 222	2437 862 544	-2.4 1.5 2.4	-1.249 0.601 1.29	2.296E-05 1.532E-02 5.044E-06	pili pili PAI-IV/group 2 capsul
	flhD ydeQ LF82_p469	Transcriptional activator flhD fimbrial-like protein ydeQ orf	350 914 1166	1331 113 333	5144 250 773	-3.9 2.2 2.3	-1.95 1.148 1.22	6.999E-08 5.470E-04 8.374E-07	pili pili PAI-IV/group 2 capsul

Table S3 : Bacterial strains and plasmids used in this study 1

 S3

	2
	Strain	or	Relevant characteristics	Reference
	plasmid
	LF82		AIEC reference strain	9
	LF82ΔeutB		LF82 isogenic mutant deleted for the eutB gene	this study
	LF82ΔeutE		LF82 isogenic mutant deleted for the eutE gene	this study
	LF82ΔeutB/peutB LF82ΔeutB containing plasmid pBK-CMV	this study
			carrying eutB
	LF82ΔcitF		LF82 isogenic mutant deleted for the citF gene	this study
	MG1655		Escherichia coli K-12 OR:H48:K-	Laboratory stock
	BW25141		Plasmid pKD4 carrying a kanamycin resistance	10
			cassette
	pKOBEG		pBAD cloning vector harboring a λ phage redγβα
			operon; chloramphenicol resistant

values shown in bold are significant.

values shown in bold are significant

coli Strain LF82. PLoS ONE 5, e12714 (2010).

Karp, P. D. et al. The EcoCyc Database. EcoSal Plus 6, (2014).

Acknowledgements

We thank Anne-Sophie Marinelli and Virginie Bonnin for technical assistance. This study was supported by the Ministère de la Recherche et de la Technologie, Inserm (U1071), INRA (USC-2018).

Competing interests

The authors declare no competing interests.