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.     

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.     

SOS regulation of the type III secretion system of enteropathogenic Escherichia coli

Genomes of bacterial pathogens contain and coordinately regulate virulence-associated genes in order to cause disease. Enteropathogenic Escherichia coli (EPEC), a major cause of watery diarrhea in infants and a model gram-negative pathogen, expresses a type III secretion system (TTSS) that is encoded by the locus of enterocyte effacement (LEE) and is necessary for causing attaching and effacing intestinal lesions. Effector proteins encoded by the LEE and in cryptic prophage are injected into the host cell cytoplasm by the TTTS apparatus, ultimately leading to diarrhea. The LEE is comprised of multiple polycistronic operons, most of which are controlled by the global, positive regulator Ler. Here we demonstrated that the LEE2 and LEE3 operons also responded to SOS signaling and that this regulation was LexA dependent. As determined by a DNase I protection assay, purified LexA protein bound in vitro to a predicted SOS box located in the divergent, overlapping LEE2/LEE3 promoters. Expression of the lexA1 allele, encoding an uncleavable LexA protein in EPEC, resulted in reduced secretion, particularly in the absence of the Ler regulator. Finally, we obtained evidence that the cryptic phage-located nleA gene encoding an effector molecule is SOS regulated. Thus, we demonstrated, for the first time to our knowledge, that genes encoding components of a TTSS are regulated by the SOS response, and our data might explain how a subset of EPEC effector proteins, encoded in cryptic prophages, are coordinately regulated with the LEE-encoded TTSS necessary for their translocation into host cells.