Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by Ler

Abstract

SummaryEnteropathogenic Escherichia coli (EPEC) produces attaching and effacing lesions (AE) on epithelial cells. The genes involved in the formation of the AE lesions are contained within a pathogenicity island named the locus of enterocyte effacement (LEE). The LEE comprises 41 open reading frames organized in five major operons: LEE1, LEE2, LEE3, LEE4 and tir. The first gene of the LEE1 operon encodes a transcription activator of the other LEE operons that is called the LEE-encoded regulator (Ler). The LEE2 and LEE3 operons are divergently transcribed with overlapping 210 promoter regions, and gene fusion studies have shown that they are both activated by Ler. Deletion analysis, using lacZ reporter fusions, of the LEE2 and LEE3 promoters demonstrated that deletions extending closer to the LEE2 transcription start site than 2247 bp lead to loss of activation by Ler, whereas only 70 bp upstream of the LEE3 transcription start site is required for Ler-mediated activation. We have purified Ler as a His-tagged protein and used it to perform DNA-binding assays with LEE2 and LEE3. We observed that Ler bound to a DNA fragment containing the 2300 to 11 region of LEE2; however, it failed to bind to a DNA fragment containing the 2300 to 11 region of LEE3, suggesting that Ler activates both operons by only binding to the regulatory region upstream of LEE2. The Ler-activatable LEE3::lacZ fusions extended to what would be 2246 bp of the LEE2 operon. A lacZ fusion from the 2300 to 11 region of LEE3 failed to be activated by Ler, consistent with our hypothesis that Ler activates the expression of LEE2 and LEE3 by binding to a region located downstream of the LEE3 transcription start site. DNase I footprinting revealed that Ler protected a region of 121 bp upstream of LEE2. Purified Ler mutated in the coiled-coil domain was unable to activate transcription and to bind to the LEE2 regulatory region. These data indicate that Ler may bind as a multimer to LEE2 and activate both divergent operons by a novel mechanism potentially involving changes in the DNA structure.

Thermoregulated expression of virulence genes in enteropathogenic Escherichia coli

“…The preferential binding of H-NS to the LEE1 and LEE2-LEE3 promoter regions supports the genetic data that H-NS either alone or together with other factors represses these operons. In the case of the LEE2-LEE3, the H-NS binding region overlaps with the Ler-binding region (Sperandio et al, 2000). The pattern of H-NS binding to the tested DNA fragments indicates that multiple copies of H-NS bind to the LEE1 and LEE2-LEE3 promoter regions in a cooperative way, typical to H-NS binding in other systems (Atlung & Ingmer, 1997).…”

“…is activated by Ler (LEE-encoded regulator), a distant homologue of H-NS, encoded by the LEE1 operon (Friedberg et al, 1999 ;Mellies et al, 1999 ;SanchezSanMartin et al, 2001 ;Sperandio et al, 2000). Therefore, the decision whether or not to activate expression of LEE1 is critical for the initiation of a regulatory cascade leading to expression of other LEE operons.…”

Ler of pathogenic Escherichia coli forms toroidal protein–DNA complexes

“…The Ler protein was expressed from pVS45 (Table 1), a derivative of the parent plasmid pBADMycHisA (Invitrogen) in the E. coli laboratory strain DH5a [supE44 D(argFlac)U169 (Q80dlacD(Z)M15) deoR hsdR17 recA1 endA1 gyrA96 thi-1 relA1]. Ler expression was induced by the addition of arabinose, and the protein was purified by nickel chromatography, performed as described by Sperandio et al (2000). In a parallel approach, the E. coli gene encoding H-NS was amplified by PCR with primers 59-hns and 39-hns (Table 2), using genomic DNA from strain MG1655 (F 2 l 2 ilv rfb-50 rph-1).…”

“…Ler relieves silencing by binding with higher affinity than H-NS (Umanski et al, 2002) to the same region upstream of LEE5, disrupting H-NS-containing nucleoprotein complexes. The LEE2 operon, encoding membrane components of the T3SS, is controlled by a similar mechanism Sperandio et al, 2000).…”

Integration host factor (IHF) mediates repression of flagella in enteropathogenic and enterohaemorrhagic Escherichia coli

“…The LEE-encoded TTSS mediates the injection of virulence factors into infected mammalian cells. Most of the LEE operons are positively regulated by the Ler regulator, which is encoded by the LEE1 operon (Friedberg et al, 1999;Mellies et al, 1999;Sperandio et al, 2000;Sanchez-SanMartin et al, 2001). Integration host factor (IHF) directly activates the expression of Ler (Friedberg et, al., 1999).…”