Transcriptional Regulation of the LEE Region

Chemical signaling plays an important role in LEE and flagella expression (Sperandio et al. 1999; Sperandio et al. 2001). The bacterial signal, autoinducer-3 (AI-3), produced by the human intestinal microbial flora, the epinephrine and nor-epinephrine host stress hormones, as well as the flagella regulon activate expression of the LEE genes (Sperandio et al. 2003). These signals act agonistically to increase LEE gene expression (Walters and Sperandio 2006) Fig. 12.3.

In addition to the regulation via stress hormones, other transcription factors are involved on LEE regulation. Iyoda and Watanabe (2004) have observed that EHEC encodes the genes pchA, pchB, and pchC (PerC homologs) that positively activate the expression of the LEE genes (Iyoda and Watanabe 2004). LEE1 encodes for Ler, the LEE-encoded regulator, which was shown to be required for the expression of all genes within the LEE (Bustamante et al. 2001; Elliott et al. 2000; Kaper et al. 2004; Mellies et al. 1999). Another important factor in the regulation of the LEE is the integration-host factor, or IHF. IHF has been shown to be required for the expression of the entire LEE through the direct activation of ler transcription (Friedberg et al. 1999). Additionally, EtrA and EivF are two negative regulators of the LEE region, acting possibly through ler transcriptional repression. The etrA and eivF genes are found within a second pathogenicity island that encodes a cryptic type III secretion system (ETT2) (Zhang et al. 2004). The histone-like nucleoid-structuring protein, H-NS, is responsible for the repression of LEE2, LEE3, and LEE5 transcription in the absence of Ler (Bustamante et al. 2001; Haack et al. 2003). RpoS, a sta-

Fig. 12.3 Model for regulation of autoinducer (AI)-3, epinephrine, and norepinephrine (NE) bind the bacterial membrane receptor QseC, which results in its autophosphorylation. QseC then phos-phorylates its response regulator QseB and initiates a complex phosphorelay signaling cascade that activates the expression of a second two-component system (QseEF), the locus of enterocyte effacement (LEE) genes, which encode various proteins, including the components of a type III section system that are involved in attaching and effacing (AE) lesion formation, the motility genes (flhDC), and Shiga toxin (stxAB). The QseEF two-component system is also involved in the expression of the LEE genes, and although its activators have not yet been elucidated, it is possible that it senses epinephrine and/or NE (Hughes and Sperandio 2008)

Fig. 12.3 Model for regulation of autoinducer (AI)-3, epinephrine, and norepinephrine (NE) bind the bacterial membrane receptor QseC, which results in its autophosphorylation. QseC then phos-phorylates its response regulator QseB and initiates a complex phosphorelay signaling cascade that activates the expression of a second two-component system (QseEF), the locus of enterocyte effacement (LEE) genes, which encode various proteins, including the components of a type III section system that are involved in attaching and effacing (AE) lesion formation, the motility genes (flhDC), and Shiga toxin (stxAB). The QseEF two-component system is also involved in the expression of the LEE genes, and although its activators have not yet been elucidated, it is possible that it senses epinephrine and/or NE (Hughes and Sperandio 2008)

tionary phase sigma factor, activates the transcription of the LEE3 operon (Sperandio et al. 1999). Finally, Hha has been reported to repress the transcription of the LEE4 operon (Sharma and Zuerner 2004).

Two previously uncharacterized genes in the LEE region, orf10 and orf11, were recently renamed GrlR, global regulator of LEE repressor, and GrlA, global regulator of LEE activator (Barba et al. 2005; Deng et al. 2004). This study suggested that GrlA is responsible for the transcriptional activation of ler, while GrlR represses ler. Additionally, it is known that Ler activates the transcription of grlRA (Barba et al. 2005; Elliott et al. 2000) and that GrlRA activates the expression of LEE2 and LEE4, independently of Ler (Russell et al. 2007).

12.3 Quorum Sensing in EHEC

Surette and Bassler reported in 1998 quorum sensing signaling in E coli K12 and S. enterica Typhimurium (Surette and Bassler 1998) through the production of autoinducer-2 (AI-2) by these bacteria (Surette et al. 1999). A common gene on these bacterial species was cloned and identified as responsible for AI-2 production, and it was named luxS (Surette et al. 1999). Later on, LuxS was characterized as the enzyme involved in the metabolism of S-adenosylmethionine and shown to convert ribose-homocysteine into homocysteine and 4,5-dihydroxy-2,3-pen-tanedione, which is the precursor of AI-2 (Schauder et al. 2001). Currently, the autoinducer referred to as AI-2 is a furanosylborate-diester in Vibrio harveyi (Chen et al. 2002), and a 2R, 4S-2-methyl-2,3,3,4-tetrahydrofuran (R-THMF) for Salmonella sp.(Miller et al. 2004).

However, at that time, no phenotypes have been shown to be regulated by quorum sensing in E. coli and Salmonella sp. In 1999, Sperandio et al. (1999) described that quorum sensing signaling activated transcription of the LEE genes and type three secretion in pathogenic E. coli, both in EHEC and in enteropatho-genic E. coli (EPEC). It was then proposed that signaling through AI-2 might be involved in virulence gene regulation in E. coli. In 2001, quorum sensing was shown to be a global regulatory mechanism in EHEC, involved in AE lesion formation, motility, metabolism, growth, and Shiga toxin expression (Sperandio et al. 2001). The EHEC's quorum sensing regulatory cascade started to be unraveled with the description of the first three quorum sensing regulators (Sperandio et al. 2002a; Sperandio et al. 2002b), which were named Quorum sensing E. coli, Qse regulators. QseA was reported as a transcription factor from the widespread LysR family, directly involved in activation of LEE1 (ler) transcription and EHEC's pathogenesis (Russell et al. 2007; Sharp and Sperandio 2007; Sperandio et al. 2002a). The QseBC regulatory system was described as a novel two-component system involved in quorum sensing regulation of flagella expression and motility (Clarke and Sperandio 2005b; Sperandio et al. 2002b).

A year later, it was shown that AI-2 was not the autoinducer involved in EHEC virulence regulation. The signal identified was a yet undescribed autoinducer, which was named autoinducer 3 (AI-3). In addition, it was reported that the eukary-otic hormones epinephrine and/or norepinephrine could substitute for the bacterial AI-3 signal to activate expression of the EHEC virulence genes. Among them, ler, the master regulator of LEE, was shown to be activated in presence of AI-3. Moreover, flagella motility of EHEC regulated via QseBC system was demonstrated to be affected by AI-3. Given that eukaryotic cell-to-cell signaling occurs through hormones, like epinephrine and norepinephrine, this cross talk between bacteria and host seem to happen through cross-signaling between quorum sensing signals and host hormones (Sperandio et al. 2003).

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