The GrlR-GrlA regulatory system coordinately controls the expression of flagellar and LEE-encoded type III protein secretion systems in enterohemorrhagic Escherichia coli

The gene function of the locus of enterocyte effacement (LEE) is essential for full virulence of enterohemorrhagic Escherichia coli (EHEC). Strict control of LEE gene expression is mediated by the coordinated activities of several regulatory elements. We previously reported that the ClpX/ClpP protease positively controls LEE expression by down-regulating intracellular levels of GrlR, a negative regulator of LEE gene expression. We further revealed that the negative effect of GrlR on LEE expression was mediated through GrlA, a positive regulator of LEE expression. In this study, we found that the FliC protein, a major component of flagellar filament, was overproduced in clpXP mutant EHEC, as previously reported for Salmonella. We further found that FliC expression was reduced in a clpXP grlR double mutant. To determine the mediators of this phenotype, FliC protein levels in wild-type, grlR, grlA, and grlR grlA strains were compared. Steady-state levels of FliC protein were reduced only in the grlR mutant, suggesting that positive regulation of FliC expression by GrlR is mediated by GrlA. Correspondingly, cell motility was also reduced in the grlR mutant, but not in the grlA or grlR grlA mutant. Because overexpression of grlA from a multicopy plasmid strongly represses the FliC level, as well as cell motility, we conclude that GrlA acts as a negative regulator of flagellar-gene expression. The fact that an EHEC strain constitutively expressing FlhD/FlhC cannot adhere to HeLa cells leads us to hypothesize that GrlA-dependent repression of the flagellar regulon is important for efficient cell adhesion of EHEC to host cells.     

Regulation of nleA in Shiga toxin-producing Escherichia coli O84:H4 strain 4795/97

Many Shiga toxin-producing Escherichia coli (STEC) strains express a type III secretion system (TTSS) encoded by the locus of enterocyte effacement (LEE). Using the TTSS, STEC is able to inject effector proteins directly into eukaryotic host cells, where they cause characteristic attaching and effacing (A/E) lesions. In addition to the LEE-encoded effectors, a number of non-LEE-encoded effectors, located on phage-associated elements, have been described. One of them, the non-LEE-encoded effector A (NleA), is widely distributed among pathogenic E. coli. In this study, we investigated the influence of environmental conditions on the expression of the phage-encoded effector nleA gene (designated nleA(4795)) present in STEC O84:H4 strain 4795/97. We demonstrated that a particular NaCl concentration and starvation stress increase the activity of the nleA(4795) promoter. Moreover, several regulators that control nleA(4795) expression were identified. The involvement of the LEE regulators Ler, GrlA, and GrlR show that nleA(4795) is integrated in the LEE regulation circuit. Furthermore, the binding of Ler to sequences upstream of nleA(4795) underlined these findings.     

PerC and GrlA independently regulate Ler expression in enteropathogenic Escherichia coli

Ler, encoded by the locus of enterocyte effacement (LEE) of attaching and effacing (A/E) pathogens, induces the expression of LEE genes by counteracting the silencing exerted by H-NS. Ler expression is modulated by several global regulators, and is activated by GrlA, which is also LEE-encoded. Typical enteropathogenic Escherichia coli (EPEC) strains contain the EAF plasmid, which carries the perABC locus encoding PerC. The precise role of PerC in EPEC virulence gene regulation has remained unclear, mainly because EPEC strains lacking the pEAF still express the LEE genes and because PerC is not present in other A/E pathogens such as Citrobacter rodentium. Here, we describe that either PerC or GrlA can independently activate ler expression and, in consequence, of LEE genes depending on the growth conditions. Both PerC and GrlA, with the aid of IHF, counteract the repression exerted by H-NS on ler and can also further increase its activity. Our results substantiate the role of PerC and GrlA in EPEC virulence gene regulation and suggest that these convergent regulatory mechanisms may have represented an evolutionary adaptation in EPEC to co-ordinate the expression of plasmid- and chromosome-encoded virulence factors needed to successfully colonize its intestinal niche.     

Structure of GrlR and the implication of its EDED motif in mediating the regulation of type III secretion system in EHEC

Enterohemorrhagic Escherichia coli (EHEC) is a common cause of severe hemorrhagic colitis. EHEC's virulence is dependent upon a type III secretion system (TTSS) encoded by 41 genes. These genes are organized in several operons clustered in the locus of enterocyte effacement. Most of the locus of enterocyte effacement genes, including grlA and grlR, are positively regulated by Ler, and Ler expression is positively and negatively modulated by GrlA and GrlR, respectively. However, the molecular basis for the GrlA and GrlR activity is still elusive. We have determined the crystal structure of GrlR at 1.9 A resolution. It consists of a typical beta-barrel fold with eight beta-strands containing an internal hydrophobic cavity and a plug-like loop on one side of the barrel. Strong hydrophobic interactions between the two beta-barrels maintain the dimeric architecture of GrlR. Furthermore, a unique surface-exposed EDED (Glu-Asp-Glu-Asp) motif is identified to be critical for GrlA-GrlR interaction and for the repressive activity of GrlR. This study contributes a novel molecular insight into the mechanism of GrlR function.