**4. Regulation of virulence genes by gastrointestinal cues**

To be fully pathogenic, bacteria must not only survive in the human GI tract but also coordi‐ nate expression of virulence determinants in response to localized gut microenvironments. An increased number of *in vitro* or *in vivo* studies have shown that both ETEC and EHEC are able to respond to various GI cues and employ these cues to modulate the expression of their virulence factors [33, 53], as described below (**Figure 2**). Compared to ETEC, where all

**Figure 2.** The figure provides a state of the art on the effects of biotic and abiotic parameters of the human gut on ETEC and EHEC virulence, as assessed by *in vitro* studies. Data related to ETEC and EHEC are surrounded by light grey and dark grey, respectively. A/E: Attaching and effacing; AI: autoinducer; CFA: colonization factor antigen; CS: *coli* surface; EA: ethanolamine; *elt*: heat‐labile enterotoxin encoding gene; *est*: heat‐stable enterotoxin encoding gene; *etpA*: ETEC two‐partner protein A encoding gene; GM1: monosialoganglioside receptor; *IhA*: IrgA homologe adhesion encoding gene; LEE: locus for enterocyte effacement; *lpf*: long polar fimbriae encoding gene; LT: heat‐labile enterotoxins; NO: nitric oxide; SCFA: short‐chain fatty acids; Stx: Shiga toxin; T3SS: type 3 secretion system.

the available studies have been performed in simple *in vitro* digestive conditions, recent data have been obtained for EHEC in more physiological conditions simulated by dynamic multi‐ compartmental models.

## **4.1. Regulation by physicochemical parameters of the human gut**

### *4.1.1. pH*

by food matrices [47–49] and age conditions [48]. In particular, thanks to the potential of the TIM model, Roussel et al. [49] have shown that differences in digestive physicochemi‐ cal parameters related to age conditions may partly explain the higher susceptibility of chil‐ dren (compared to adults) to EHEC infections and HUS. Additional studies performed under human‐simulated colonic conditions (including colonic microbiota) have shown that EHEC strains were not able to colonize [50], probably due to the barrier effect of gut microbiota or to the high short‐chain fatty acid (SCFAs) concentrations found in the colon and known to inhibit EHEC growth [51, 52]. Taken together, these data suggest that the ability of EHEC to colonize the human gut would be rather linked to growth renewal of the pathogen in the

To be fully pathogenic, bacteria must not only survive in the human GI tract but also coordi‐ nate expression of virulence determinants in response to localized gut microenvironments. An increased number of *in vitro* or *in vivo* studies have shown that both ETEC and EHEC are able to respond to various GI cues and employ these cues to modulate the expression of their virulence factors [33, 53], as described below (**Figure 2**). Compared to ETEC, where all

**Figure 2.** The figure provides a state of the art on the effects of biotic and abiotic parameters of the human gut on ETEC and EHEC virulence, as assessed by *in vitro* studies. Data related to ETEC and EHEC are surrounded by light grey and dark grey, respectively. A/E: Attaching and effacing; AI: autoinducer; CFA: colonization factor antigen; CS: *coli* surface; EA: ethanolamine; *elt*: heat‐labile enterotoxin encoding gene; *est*: heat‐stable enterotoxin encoding gene; *etpA*: ETEC two‐partner protein A encoding gene; GM1: monosialoganglioside receptor; *IhA*: IrgA homologe adhesion encoding gene; LEE: locus for enterocyte effacement; *lpf*: long polar fimbriae encoding gene; LT: heat‐labile enterotoxins; NO: nitric

oxide; SCFA: short‐chain fatty acids; Stx: Shiga toxin; T3SS: type 3 secretion system.

distal parts of the small intestine than the ability to maintain in the colon.

10 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

**4. Regulation of virulence genes by gastrointestinal cues**

Once ingested, pathogens are exposed to the host digestive tract characterized by acid con‐ ditions in the stomach where pH gradually decreases during digestion from around 6 to 2, followed by pH close to neutrality in the small intestine.

For ETEC, the release of ST seems to be not pH‐dependent [54], while it is acknowledged that extracellular pH has an influence on the release of LT toxin which increases with alkalinity [55, 56]. ETEC seems to use the pH gradient in the GI tract to modulate LT toxin production and secretion: when bacteria reach the small intestine, alkaline pH induces both transcription and maximal release of LT [42].

For EHEC, House et al. [57] have examined, using DNA microarrays, the gene expression profiles of EHEC O157 that had been acid stressed and then neutralized relative to the same unstressed strain. Virulence factors associated with adhesion, motility, and type III secretion were significantly modulated leading to enhancement of motility and host cell adhesion. The T3SS genes encoding proteins that mediate colonization and infection in the large intestine were downregulated following acid stress [33, 57]. Impact of low pH on Stx gene expression and production is not yet fully understood: House et al. [57] have shown no change whereas other studies have revealed that acid pH decreases Stx production [58, 59]. In the gastric and small intestinal TIM model, Roussel et al. [49] have shown that *stx1* and *stx2* genes were upregulated in the gastric compartment even if Stx‐mediated cytotoxicity is generally associ‐ ated with distal parts of the small intestine or large intestine. Higher expression levels were observed under child digestive conditions compared to adult ones where less acidic condi‐ tions are found, which is in accordance with the results of Yuk et al. [58] and Huang et al. [59].

### *4.1.2. Bile*

Once the small intestine is reached, bile salts form a major challenge to pathogens, with bile concentrations sequentially decreasing from duodenum to colon due to reabsorption.

Chatterjee and Chowdhury [60] have shown *in vitro* that 2 g/L crude bile can prevent the binding of LT toxin to GM1 and that this effect was associated to arachidonic, linoleic, and oleic unsaturated fatty acids detected in crude bile. The same authors demonstrated *in vivo* in rabbit ileal loops that linoleic acid prevented LT‐mediated fluid accumulation in a dose‐ dependent manner [60]. In another study by Nicklasson et al. [61], 1.5 g/L crude bile and 2 g/L bile salts sodium deoxycholate and sodium glycocholate‐induced *in vitro* the expression of CS5‐encoding gene *csfD*. A global transcriptional analysis of two ETEC strains showed that bile salts at a concentration of 30 g/L in LB medium upregulated *estA*, *eltA,* or *etpA* (encoding for STa, LTa enterotoxins, and EtpA, respectively) while *csoA* and *cstA* (encoding for CS1 and CS3 colonization factors) were downregulated [43]. In this study, the transcriptional response to bile salts was strain‐dependent, suggesting that the results should not be extrapolated to the entire pathovar without further investigation. Finally, at the protein level, 1.5 g/L bile salts were required for surface expression of at least CS5, CS7, CS8, CS12, CS14, CS17, and CS19 [62–64]. Haines et al. [62] have shown that bile salts seem not to be required for the expres‐ sion of CS1, CS2, and CS3, while the opposite was demonstrated by Sjoling et al. [63]. These results suggest that both interaction of LT toxin with its receptor and expression of ETEC colonization factors may be differentially induced along the human intestine where bile acid concentrations range from 2 to 20 g/L.

effector proteins were increased under microaerobic conditions (1–2% oxygen). Microaerobiosis also significantly reduced bacterial growth as well as Stx production and release into the medium, while Stx translocation across the epithelial monolayer was enhanced. The role of oxygen levels on modulation of EHEC virulence was further confirmed by Lewis et al. [27] who showed in *in vitro* organ culture (IVOC) of human colonic biopsy samples that A/E lesion formation was dependent on oxygen levels. These lesions were suppressed under oxygen‐ rich culture conditions routinely used for IVOC. Taken together, these results suggest that the microaerobic environment adjacent to the intestinal microvilli may upregulate the expression

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Fluid shear can be defined as distribution of frictional forces due to the hydrodynamic flow generated by GI peristaltic activity against the surface of intestinal epithelial cells. In the human gut, there is a decreasing gradient of fluid shear stress from mucosa to gut lumen. It has been generally assumed that shear stress inhibits pathogen adhesion, thereby serving as a non‐specific host defense against bacterial colonization [72]. For both ETEC and EHEC, this

Tchesnokova et al. [72] have shown, using *in vitro* erythrocytes and Caco‐2 cell models, a shear‐enhanced binding of intestinal CfaE, the tip‐localized minor subunit of CFA/I, in both prototypical and clinical ETEC strains. EHEC attachment to host cells is also enhanced by levels of shear force similar to peristaltic forces in the intestinal tract, which are required to fully activate LEE‐encoded virulence mechanisms [73]. These preliminary data suggest that, in addition to a range of chemical environmental signals, ETEC and EHEC are capable of

During passage through the human gut, enteric pathogenic bacteria such as ETEC and EHEC also have to face a high number of commensal bacteria that compete with them for nutrients and space. There is scarce data on the interactions of EHEC, but even more so for ETEC, with

For ETEC, only two studies have investigated gut microbiota changes during ETEC challenge [74, 75]. The authors conclude that ETEC infections are associated with a rapid and reversible change in gut microbial community structure as well as a significant decrease in overall bacte‐ ria diversity. However, there is no available data on how gut microbiota may influence ETEC

With regard to EHEC, Thévenot et al. [50] have recently shown in an *in vitro* model of the human colon, that *E. coli* O157:H7 has an individual dependent effect on the colonic micro‐

of EHEC virulence factors that promote successful colonization of the large intestine.

concept has been very poorly described in the literature.

**4.2. Regulation by biotic factors of the human gut**

*4.2.1. Gut microbiota and their metabolites*

*4.2.1.1. Gut microbiota*

human gut microbiota.

virulence.

sensing and responding to mechanical cues in the human GI tract.

*4.1.5. Fluid shear*

Studies have also shown that bile may serve as an environmental cue for EHEC by modu‐ lating the expression of specific virulence factors [33]. DNA microarray analysis of EHEC O157:H7 treated with 1.5 g/L bile salts showed upregulation of *acrA* and *acrB* genes encoding a bile salts efflux pump [65]. Expression of several other well‐known virulence factors includ‐ ing those encoded on the LEE pathogenicity island, was not altered by bile salt treatment. On the contrary, a significant decrease in *eae* gene transcripts was observed *in vitro* by other authors when 5–8 g/L bile salts were added [66, 67]. Bile salts also modulate the expression of other adhesins, such as Lpf: Arenas‐Hernández et al. [68] and Yin et al. [66] found that con‐ centrations of 1.5–5 g/L led to an upregulation of *lpf* genes. In the TIM system, *eae* and *lpf* over‐ expression occurred under child digestive conditions at the end of *in vitro* digestion, when most of the bacterial cells have reached the distal parts of the small intestine [49]. This might suggest a higher ability of EHEC to colonize the terminal ileum or colon in children compared to adults. Lastly, there is no consensus for the effect of bile salts on *stx* gene expression. Kus et al. [65] reported that 1.5 g/L bile salt downregulated *stx2* genes, whereas no influence was observed by Hamner et al. [67] with concentrations of 8 g/L.

### *4.1.3. Digestive enzymes*

Very few studies have investigated how human digestive enzymes may influence the expres‐ sion of virulence genes in pathogens, none in EHEC and only two in ETEC. In the latter, *in vitro* studies have shown that trypsin, an endopeptidase secreted by duodenal epithelial cells, is able to increase LT release [55] and its secretory activity [69].

### *4.1.4. Oxygen levels*

Various oxygen levels can be found in the human GI tract with concentrations decreasing from the upper to the lower digestive tract and from mucosal surfaces to gut lumen. Up to date, the effect of various oxygen concentrations on pathogen virulence has been studied only in EHEC.

In an *in vitro* cell culture model, James and Keevil [70] have shown that the presence of oxygen enhanced EHEC ability to adhere to epithelial cells. In other recent studies, polarized human colon carcinoma cells in a vertical diffusion chamber system were used to investigate the influ‐ ence of reduced apical oxygen levels on EHEC colonization [38] and Stx production [71]. The authors demonstrated that both EHEC‐host adhesion and expression and translocation of T3SS effector proteins were increased under microaerobic conditions (1–2% oxygen). Microaerobiosis also significantly reduced bacterial growth as well as Stx production and release into the medium, while Stx translocation across the epithelial monolayer was enhanced. The role of oxygen levels on modulation of EHEC virulence was further confirmed by Lewis et al. [27] who showed in *in vitro* organ culture (IVOC) of human colonic biopsy samples that A/E lesion formation was dependent on oxygen levels. These lesions were suppressed under oxygen‐ rich culture conditions routinely used for IVOC. Taken together, these results suggest that the microaerobic environment adjacent to the intestinal microvilli may upregulate the expression of EHEC virulence factors that promote successful colonization of the large intestine.

## *4.1.5. Fluid shear*

CS3 colonization factors) were downregulated [43]. In this study, the transcriptional response to bile salts was strain‐dependent, suggesting that the results should not be extrapolated to the entire pathovar without further investigation. Finally, at the protein level, 1.5 g/L bile salts were required for surface expression of at least CS5, CS7, CS8, CS12, CS14, CS17, and CS19 [62–64]. Haines et al. [62] have shown that bile salts seem not to be required for the expres‐ sion of CS1, CS2, and CS3, while the opposite was demonstrated by Sjoling et al. [63]. These results suggest that both interaction of LT toxin with its receptor and expression of ETEC colonization factors may be differentially induced along the human intestine where bile acid

12 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

Studies have also shown that bile may serve as an environmental cue for EHEC by modu‐ lating the expression of specific virulence factors [33]. DNA microarray analysis of EHEC O157:H7 treated with 1.5 g/L bile salts showed upregulation of *acrA* and *acrB* genes encoding a bile salts efflux pump [65]. Expression of several other well‐known virulence factors includ‐ ing those encoded on the LEE pathogenicity island, was not altered by bile salt treatment. On the contrary, a significant decrease in *eae* gene transcripts was observed *in vitro* by other authors when 5–8 g/L bile salts were added [66, 67]. Bile salts also modulate the expression of other adhesins, such as Lpf: Arenas‐Hernández et al. [68] and Yin et al. [66] found that con‐ centrations of 1.5–5 g/L led to an upregulation of *lpf* genes. In the TIM system, *eae* and *lpf* over‐ expression occurred under child digestive conditions at the end of *in vitro* digestion, when most of the bacterial cells have reached the distal parts of the small intestine [49]. This might suggest a higher ability of EHEC to colonize the terminal ileum or colon in children compared to adults. Lastly, there is no consensus for the effect of bile salts on *stx* gene expression. Kus et al. [65] reported that 1.5 g/L bile salt downregulated *stx2* genes, whereas no influence was

Very few studies have investigated how human digestive enzymes may influence the expres‐ sion of virulence genes in pathogens, none in EHEC and only two in ETEC. In the latter, *in vitro* studies have shown that trypsin, an endopeptidase secreted by duodenal epithelial

Various oxygen levels can be found in the human GI tract with concentrations decreasing from the upper to the lower digestive tract and from mucosal surfaces to gut lumen. Up to date, the effect of various oxygen concentrations on pathogen virulence has been studied only

In an *in vitro* cell culture model, James and Keevil [70] have shown that the presence of oxygen enhanced EHEC ability to adhere to epithelial cells. In other recent studies, polarized human colon carcinoma cells in a vertical diffusion chamber system were used to investigate the influ‐ ence of reduced apical oxygen levels on EHEC colonization [38] and Stx production [71]. The authors demonstrated that both EHEC‐host adhesion and expression and translocation of T3SS

concentrations range from 2 to 20 g/L.

*4.1.3. Digestive enzymes*

*4.1.4. Oxygen levels*

in EHEC.

observed by Hamner et al. [67] with concentrations of 8 g/L.

cells, is able to increase LT release [55] and its secretory activity [69].

Fluid shear can be defined as distribution of frictional forces due to the hydrodynamic flow generated by GI peristaltic activity against the surface of intestinal epithelial cells. In the human gut, there is a decreasing gradient of fluid shear stress from mucosa to gut lumen. It has been generally assumed that shear stress inhibits pathogen adhesion, thereby serving as a non‐specific host defense against bacterial colonization [72]. For both ETEC and EHEC, this concept has been very poorly described in the literature.

Tchesnokova et al. [72] have shown, using *in vitro* erythrocytes and Caco‐2 cell models, a shear‐enhanced binding of intestinal CfaE, the tip‐localized minor subunit of CFA/I, in both prototypical and clinical ETEC strains. EHEC attachment to host cells is also enhanced by levels of shear force similar to peristaltic forces in the intestinal tract, which are required to fully activate LEE‐encoded virulence mechanisms [73]. These preliminary data suggest that, in addition to a range of chemical environmental signals, ETEC and EHEC are capable of sensing and responding to mechanical cues in the human GI tract.

### **4.2. Regulation by biotic factors of the human gut**

## *4.2.1. Gut microbiota and their metabolites*

### *4.2.1.1. Gut microbiota*

During passage through the human gut, enteric pathogenic bacteria such as ETEC and EHEC also have to face a high number of commensal bacteria that compete with them for nutrients and space. There is scarce data on the interactions of EHEC, but even more so for ETEC, with human gut microbiota.

For ETEC, only two studies have investigated gut microbiota changes during ETEC challenge [74, 75]. The authors conclude that ETEC infections are associated with a rapid and reversible change in gut microbial community structure as well as a significant decrease in overall bacte‐ ria diversity. However, there is no available data on how gut microbiota may influence ETEC virulence.

With regard to EHEC, Thévenot et al. [50] have recently shown in an *in vitro* model of the human colon, that *E. coli* O157:H7 has an individual dependent effect on the colonic micro‐

biota, as assessed by qPCR analysis on major phyla and genus. The same authors also showed that EHEC infection led in the *in vitro* colonic environment to a significant increase in *stx1*, *stx2,* and *eae* expression 9–12 h post‐administration. Besides, it has been also proposed that EHEC was sensing autoinducers produced by the GI microbiota, such as the quorum signaling molecule AI‐3. EHEC respond to AI‐3 by increasing flagellar synthesis and motility that allow the pathogen to more closely approach the mucosal epithelium at the site of colonization [76]. On the contrary, other soluble factors secreted by the normal gut microbiota may protect the host against EHEC infection. De Sablet et al. [77] have shown, in cecal contents of gnotobiotic rats colonized with human microbiota, that small molecules produced in part by *Bacteroides thetaiotaomicron*, a predominant species of the normal human intestinal microbiota, repressed *stx2* mRNA expression. Mutants of *B. thetaiotaomicron* with impaired production of a specific transporter of vitamin B12 were no longer able to inhibit the production of Stx2 [78]. This work suggests that concentration of vitamin B12 in the gut and by extension, activities of commensal bacterial species producing and/or consuming vitamin B12, may modulate the production of the main virulence factor of EHEC. Other studies have also demonstrated that the interplay between the nutrient requirements of normal flora and EHEC is important in determining pathogen virulence [76]. Njoroge et al. [79] uncovered the importance of glucose availability in regulating T3SS by EHEC: high‐glucose growth media suppressed type III secretion while low‐glucose conditions induced LEE expression. EHEC also use fucose that is made available from mucus by the microbiota (especially by *Bacteroides thetaiotaomicron*) to modulate their own metabolism and virulence. Pacheco et al. [80] described a novel two‐component system that enables regulation of virulence gene expression and carbon‐source choice by EHEC upon sensing fucose, resulting in a decrease in LEE transcript levels. All these results tend to indi‐ cate that differential microbiota composition may contribute to host resistance or susceptibil‐ ity to EHEC infections. Then, differences in diet and antibiotic regimens, which cause shifts in the composition of the GI microbiota may also influence the outcome of the disease.

They showed that when EHEC were exposed to SCFA mixes representative of the small intestine, there was a significant upregulation of flagellar genes, flagellar protein FliC, and motility, while the opposite was observed with SCFA mixes representative of the large intestine. Lastly, a high‐fiber diet, via enhanced butyrate levels, increased host's expression of Gb3 and susceptibility of mice to disease [86]. Conversely, increased levels of microbi‐ ota‐derived acetate protected animals from disease that is caused by the toxin. Collectively, these data suggest that molecular cues secreted by commensal microbiota such as SCFAs may modulate EHEC motility, adhesion, and toxin production, differently in the small and

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Microbial endocrinology is a newly recognized microbiology research area investigating the interactions of bacteria with stress‐associated hormones, such as catecholamine. Among these hormones, only epinephrine and norepinephrine have been investigated as environmental

Lyte et al. [87] demonstrated that physiological concentrations of norepinephrine increased the *in vitro* growth of an ETEC strain isolated from calf, as well as the expression of the viru‐ lence factor F5 fimbrial adhesin. On the contrary, Sturbelle et al. [88] did not observe any effect of norepinephrine or epinephrine on the *in vitro* growth of a piglet ETEC strain, and Haines et al. [62] found a significant inhibition of porcine ETEC growth by norepinephrine. However, a significant increase in motility and expression of F4 fimbriae and LT toxin‐encoding genes was shown in the ETEC culture supplemented with conditioned medium (containing auto‐ inducers) and epinephrine [88]. Lastly, Haines et al. [62] found that norepinephrine inhibited

As described for ETEC, Lyte et al. [89] found that norepinephrine increased *in vitro* EHEC growth. EHEC also use norepinephrine as a signal for differential regulation of virulence factors mediating invasion, motility, and A/E lesion formation [90]. Regulation of EHEC viru‐ lence by epinephrine and norepinephrine is still not fully understood but it has been shown that the pathogen uses the histidine sensor kinases QseC and QseE as sensors of the two hor‐ mones [33, 76]. So, host‐derived hormones epinephrine and/or norepinephrine seem to assist ETEC and EHEC in cueing their site of colonization and enhance approach to the epithelial

The influence of other GI factors, such as ethanolamine (EA) and nitric oxide (NO), has been studied on EHEC virulence, but not on ETEC. However, the nature of the associated regula‐

EA comes from the turnover of intestinal epithelial cells and commensal microbiota and is gen‐ erated from the breakdown of phosphatidylethanolamine. EHEC cultured in minimal media containing EA showed increased expression of both s*tx2* and genes encoded on the LEE patho‐ genicity island, as well as a higher number of attaching and effacing (A/E) lesions on host

large intestines.

*4.2.2. Host hormones*

cues for ETEC and EHEC.

CFA/I expression in an ETEC strain isolated from humans.

layer through increased motility and adhesion.

*4.2.3. Other factors*

epithelial cells [91].

tions is still not fully understood.

### *4.2.1.2. Short‐chain fatty acids*

Several studies have investigated how ETEC and EHEC may respond to gut microbiota metabolites such as SCFAs. The three main SCFAs present in the intestine are acetate, propio‐ nate, and butyrate and their concentrations vary from the small intestine to the colon.

A single study with ETEC has shown that addition of SCFAs from C‐2 to C‐7 at a con‐ centration of 2 mg/mL in the culture medium significantly reduced or even abolished LT production [81]. A higher number of studies have evaluated how EHEC may sense SCFAs. Acetate (10–40 mM) and propionate (2–10 mM) had no effect on Stx2 production levels *in vitro* [78] while acetate production by *Bifidobacterium* strains was associated with an anti‐ infectious activity through the inhibition of Stx production and translocation [82]. Low SCFA concentrations (particularly of butyrate—from 6.25 to 25 mM), more typical of the distal ileum, enhanced the expression of EHEC virulence genes involved in motility, adhe‐ sion, and induction of A/E lesion formation [51, 52]. Other studies reported that high con‐ centrations of SCFAs (above 50 mM), typically found in the distal colon, were associated with increased expression of T3SS [83] and Iha adhesin [84]. Very recently, Lackraj et al. [85] have investigated how EHEC modulate flagella expression and motility in response to SCFA mixes typical in compositions and concentrations of the small and large intestines. They showed that when EHEC were exposed to SCFA mixes representative of the small intestine, there was a significant upregulation of flagellar genes, flagellar protein FliC, and motility, while the opposite was observed with SCFA mixes representative of the large intestine. Lastly, a high‐fiber diet, via enhanced butyrate levels, increased host's expression of Gb3 and susceptibility of mice to disease [86]. Conversely, increased levels of microbi‐ ota‐derived acetate protected animals from disease that is caused by the toxin. Collectively, these data suggest that molecular cues secreted by commensal microbiota such as SCFAs may modulate EHEC motility, adhesion, and toxin production, differently in the small and large intestines.
