*4.2.2. Host hormones*

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

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

the composition of the GI microbiota may also influence the outcome of the disease.

nate, and butyrate and their concentrations vary from the small intestine to the colon.

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‐

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.

*4.2.1.2. Short‐chain fatty acids*

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 cues for ETEC and EHEC.

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

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 layer through increased motility and adhesion.

### *4.2.3. Other factors*

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‐ tions is still not fully understood.

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 epithelial cells [91].

NO is an essential mediator of the innate immune response of infected colonic mucosa. Chemical or cellular sources of NO have been shown to inhibit *stx‐* and LEE‐encoded genes mRNA expression and Stx synthesis, without altering EHEC viability [92, 93].

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