Ler Is a Negative Autoregulator of theLEE1Operon in EnteropathogenicEscherichia coli

Abstract

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. Essential for colonization of the host intestine is the LEE pathogenicity island, which comprises a cluster of operons encoding a type III secretion system and related proteins. The LEE1 operon encodes Ler, which positively regulates many EPEC virulence genes in the LEE region and elsewhere in the chromosome. We found that Ler acts as a specific autorepressor of LEE1 transcription. We further show that Ler specifically binds upstream of the LEE1 operon in vivo and in vitro. A comparison of the Ler affinities to different DNA regions suggests that the autoregulation mechanism limits the steady-state level of Ler to concentrations that are just sufficient for activation of the LEE2 and LEE3 promoters and probably other LEE promoters. This mechanism may reflect the need of EPEC to balance maximizing the colonization efficiency by increasing the expression of the virulence genes and minimizing the immune response of the host by limiting their expression. In addition, we found that the autoregulation mechanism reduces the cell-to-cell variability in the levels of LEE1 expression. Our findings point to a new negative regulatory circuit that suppresses the noise and optimizes the expression levels of ler and other LEE1 genes.Colonizing enteropathogens compete with the gut flora to gain a foothold in the host tissue by expressing powerful colonization factors. However, to reduce the immune response of the host, the pathogen should minimize the expression of the colonization factors. To resolve this dilemma, pathogens evolved regulatory mechanisms that optimize the expression levels and timing, thus maintaining expression of just enough colonization factors and only when needed. Another layer of complexity is added when the colonization is dependent on the assembly of organelles like the type III secretion systems (TTSS), which are composed of ϳ30 different proteins of various relative amounts and encoded by several operons. In these cases, an orderly expression program is required for efficient assembly of the organelle.Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. It employs the TTSS as a molecular syringe to inject a battery of toxic or colonization proteins into the membrane and cytoplasm of infected host cells (4). The TTSS and some of the effectors are encoded by a 35.6-kbp pathogenicity island, termed the locus for enterocyte effacement (LEE). The LEE consists of 41 genes, organized in five major operons (LEE1 to LEE5) and several additional transcriptional units (10,19). Ler, an H-NS paralog, encoded by the first gene of the LEE1 operon, is a key regulator of the LEE regulon, positively regulating expression of LEE2, LEE3, LEE4, LEE5, espG, and map (11,19,24,30). The regulation of ler (LEE1 operon) is complex and involves many factors, including H-NS, integration host factor (IHF), Fis, PerC, BipA, GrlA, GrlR, GadX, and quorum sensing (2,7,11,13,14,17,19,26,28,30,32). Most of these factors appe...

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

“…Conversely, the GrlA-independent expression of the ler promoter in pCRler-80 was reduced sevenfold in the presence of a plasmid expressing Ler (Fig. 2B), supporting the notion that Ler may negatively autoregulate its own expression to optimize its cellular levels, preventing the uncontrolled expression of LEE genes, as recently proposed (2). As a control, the expression of a transcriptional fusion to the LEE2 promoter (pLEE2-cat), whose expression is Ler dependent, was measured.…”

“…In this way, the active feedback loop will increase the cellular concentration of Ler, which then specifically counteracts the H-NS-mediated repression of several LEE and non-LEE promoters. To prevent the detrimental accumulation of Ler or of the proteins encoded by Ler-regu-lated genes in the cell, the Ler-GrlA feedback loop could be negatively modulated when Ler reaches the threshold concentration that represses ler transcription, as recently proposed (2). Alternatively, other elements could establish a checkpoint to prevent Ler overexpression.…”

“…GadX regulates the expression of the perABC operon and thus indirectly regulates the expression of ler (41). It has been reported that Ler binds to its own regulatory region and autorepresses its transcription in a concentration-dependent manner (2). The negative regulation of LEE gene expression is also mediated by YhiE and YhiF (44) as well as by EtrA (E. coli type III secretion system 2 regulator A) and EivF (49) by mechanisms that remain to be defined.…”

Screening for Host Proteins Interacting with Escherichia Coli O157:H7 EspF using Bimolecular Fluorescence Complementation

“…EspF thus emerges as the 'Swiss army knife' of a bacterial pathogen [11,12]. EHEC O157:H7 employs a type-III secretion system to export toxic factors and effector proteins to host cells after adhering to the brush border of epithelial cells [5,[13][14]. The production of virulence factors, subsequent invasion and diffusion, and the interaction of effector proteins with host proteins are key steps in EHEC O157:H7 pathogenesis.…”

Global Regulator of Virulence A (GrvA) Coordinates Expression of Discrete Pathogenic Mechanisms in Enterohemorrhagic Escherichia coli through Interactions with GadW-GadE

“…The master LEE regulator, Ler, encoded as the first gene of LEE1, positively stimulates LEE transcription by relieving H-NS-mediated repression. Ler further activates the LEE-encoded regulators GrlA and GrlR, which in turn activate and repress ler transcription, respectively (5)(6)(7)(8)(9)(10). Numerous non-LEE-encoded regulators also converge on ler and grlA promoters to coordinate LEE-dependent colonization with environmental/physiological cues (11)(12)(13)(14)(15)(16)(17)(18)(19).…”