A Positive Regulatory Loop Controls Expression of the Locus of Enterocyte Effacement-Encoded Regulators Ler and GrlA

Abstract

The formation of attaching and effacing (A/E) lesions on intestinal epithelial cells is an essential step in the pathogenesis of human enteropathogenic and enterohemorrhagic Escherichia coli and of the mouse pathogen Citrobacter rodentium. The genes required for the development of the A/E phenotype are located within a pathogenicity island known as the locus of enterocyte effacement (LEE). The LEE-encoded transcriptional regulators Ler, an H-NS-like protein, and GrlA, a member of a novel family of transcriptional activators, positively control the expression of the genes located in the LEE and their corresponding virulence. In this study, we used C. rodentium as a model to study the mechanisms controlling the expression of Ler and GrlA. By deletion analysis of the ler and grlRA regulatory regions and complementation experiments, negative and positive cis-acting regulatory motifs were identified that are essential for the regulation of both genes. This analysis confirmed that GrlA is required for the activation of ler, but it also showed that Ler is required for the expression of grlRA, revealing a novel regulatory loop controlling the optimal expression of virulence genes in A/E pathogens. Furthermore, our results indicate that Ler and GrlA induce the expression of each other by, at least in part, counteracting the repression mediated by H-NS. However, whereas GrlA is still required for the optimal expression of ler even in the absence of H-NS, Ler is not needed for the expression of grlRA in the absence of H-NS. This type of transcriptional positive regulatory loop represents a novel mechanism in pathogenic bacteria that is likely required to maintain an appropriate spatiotemporal transcriptional response during infection.Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium belong to a family of bacterial pathogens causing a destructive lesion of the intestinal enterocyte, called the attaching and effacing (A/E) lesion, as well as gastrointestinal disorders in infected hosts (reviewed in references 28 and 33). EPEC is an important etiological agent of childhood diarrhea in developing countries, whereas EHEC is the cause of frequent outbreaks of food and water poisoning in the developed world. In addition to causing diarrhea, an EHEC infection can result in severe complications, such as hemorrhagic colitis and hemolytic-uremic syndrome (reviewed in reference 33). Due to the specificity of EPEC and EHEC for human hosts, a corresponding small-animal infection model does not exist. Thus, most of the current models to explain EHEC and EPEC pathogen-host interactions, such as those for A/E lesion formation, have been developed based on in vitro studies performed with infected cultured epithelial cells. In recent years, C. rodentium has become accepted as a representative infection system to study the mechanisms leading to the production of the A/E lesion and A/E-associated pathogenesis (12, 13, 47).The A/E lesion is characterized by a localized loss of microvilli from ...

Differences in adherence and virulence gene expression between two outbreak strains of enterohaemorrhagic Escherichia coli O157 : H7

“…Regulation of the LEE is complex and is influenced by multiple environmental factors that act on several chromosomal genes, which in turn fine-tune LEE expression through LEE-encoded regulators, Ler, GrlA and GrlR (Deng et al, 2004;Spears et al, 2006). Once activated, Ler leads to induction of operons LEE2, LEE3, LEE5 and LEE4 (Barba et al, 2005;Haack et al, 2003). More so, GrlA can stimulate expression of Ler (Deng et al, 2004) as well as of LEE2 and LEE4 independently of Ler (Russell et al, 2007).…”

Anti-silencing: overcoming H-NS-mediated repression of transcription in Gram-negative enteric bacteria

“…The Ler DNAbinding protein is encoded by the LEE (locus of enterocyte effacement) pathogenicity island of enterohaemorrhagic E. coli (EHEC) and EPEC. It activates the transcription of the major virulence operons in the island at 37 u C by opposing the silencing activity of H-NS (Barba et al, 2005;Bustamante et al, 2001;Haack et al, 2003;Umanski et al, 2002). Ler and H-NS are partial paralogues whose oligomerization domains are highly divergent coiled-coils; there is no evidence that Ler and H-NS form heterodimers.…”

Enterohemorrhagic Escherichia coli Virulence Gene Regulation

“…4). The GrlRA regulators constitute a complicated feedback loop whereby GrlA directly activates ler expression, and GrlR acts posttranslationally to reduce the cellular quantity of the GrlA protein (28,61,64,65). Once expressed, Ler, in turn, acts as an H-NS antisilencer to increase the expression of LEE2, LEE3, LEE4, and LEE5 operons (Fig.…”

“…To demonstrate the many inputs controlling ler gene expression, researchers working with both EHEC and enteropathogenic E. coli (EPEC) found that Ler is stimulated in response to environmental signals such as temperature, pH, iron, ammonium, calcium, bicarbonate (50)(51)(52)(53)(54), and quorum-sensing signaling (55,56), as well as the proteins IHF (57), Fis (58), BipA (59), PerC homologs, or Pch (60), GrlRA (61,62), and GadX (63) (Fig. 4).…”