Multiple functions of l0036 in the regulation of the pathogenicity island of enterohaemorrhagic Escherichia coli O157:H7

Abstract

Diarrhoeagenic enterohaemorrhagic Escherichia coli and enteropathogenic E. coli attach to human intestinal epithelium and efface brush-border microvilli, forming an A/E (attaching and effacing) lesion. These human pathogens are phenotypically similar to the mouse pathogen Citrobacter rodentium. Genetically, they all have a homologous set of virulent genes involved in the A/E lesion, and these genes are organized on a LEE (locus of enterocyte effacement), a pathogenicity island. This island comprises 41 specific open reading frames, of which most are organized at five operons, LEE1, LEE2, LEE3, LEE4 and tir (LEE5). The expression of the LEE genes is regulated in a complicated manner, and current knowledge is that there are at least two positive regulators, Ler (LEE-encoded regulator) and GrlA (global regulator of LEE activator), and one negative regulator, called GrlR (global regulator of LEE repressor). In enterohaemorrhagic E. coli, GrlA is encoded by l0043, whereas GrlR is encoded by l0044. Here we report a fourth regulatory gene located in LEE3, namely l0036. Its expression is tightly controlled. When overexpressed, this factor, named Mpc (multiple point controller), interacts with Ler and suppresses the expression of the LEE proteins. When the translation is not initiated or terminated before maturation, the type III secretion of effectors is completely abolished. Therefore, together with the fact that several cis elements reside in the region that l0036 spans, l0036 appeared to have multiple functions in the regulation of LEE expression.

Figure 1. Effects on the expression and secretion of representative LEE proteins when the initiation codon of l0036 was altered

Bacteria transformed with (+) or without (−) pQL36 were cultivated in M9 medium for 6 h. Bacterial lysates and proteins from spent medium were then prepared and analysed by Western blotting using antibodies specific to the antigen indicated. Abbreviations: WT, the wild-type strain 43888; AC36, a mutant strain 43888 with initiation codon of l0036 altered from ATG to CTG.

Figure 2. LEE3 genes downstream of l0036 required for AC36 to complement the defect in the type III secretion

(A) Schematic of gene organization (not to scale) flanking l0036 and illustration of the DNA inserts in the complementation plasmids. (B) Proteins from bacterial lysates and spent medium were analysed as described above in the legend to Figure 1. O157, the antigen detected by anti-O157 antibodies from Difco.

Figure 3. Analysis of the role of l0036 in bacterial transcription and translation

(A) Illustration of the transcripts driven by T7 promoter in various plasmids. pBST7-A has the insert copied from the parental sequence; pBST7-C differs from pBST7-A by replacing the initiation codon of l0036 with CTG; pBST7-A72 differs by placing TGA at the 72nd codon; pBST7-186 has the 5′ LEE3 nucleotides deleted down to the 183rd base of the escV ORF. (B) Protein expression profiles derived from the transcripts in (A). Proteins synthesized in the plasmid-transformed bacteria were labelled with [³⁵S]methionine in the presence of both IPTG and rifampicin. Bands labelled were those presumably derived from ORFs of the transcript (see the text for the product assignment). (C) Analysis of the 5′ nucleotides and the N-terminal amino acid residues encoded by escV. All methionine residues in this region are highlighted, and the possible ribosome-binding sites are underlined. The arrowhead indicates the 5′ nucleotide of the truncated escV, to which the T7 promoter was directly linked in pBST7-186.

Figure 4. Dosage effect of L0036 on the expression and secretion of the LEE proteins

The amount of L0036 expressed in bacteria was controlled by plasmids carrying the same expression cassette but with different copy numbers. pQL36N is identical with pQL36, except that L0036 was out of frame and a stop codon was generated immediately after the N-terminal RGS-His₆ tag. Bacteria used were the wild-type strain 43888, and the protein analyses were carried out as described in the legend to Figure 1.

Figure 5. Ler is involved in the effects of L0036

(A) Counteracting effect of L0036 on the activation of Ler on the LEE expression. Effects of expressing L0036 or L0044 in addition to Ler were examined by analysis of the LEE proteins in a Ler-deleted mutant, strain 43888 ΔLer. The mutant strain receiving various plasmids were analysed for the LEE proteins expressed in the bacterial lysates as described in the legend to Figure 1. (B) Interaction of Ler and L0036 as shown by a bacterial two-hybrid system. ORFs were cloned separately into expression plasmids to serve as target protein and bait. The interactions of the target and bait proteins in a reporter strain were revealed by an increase in colony number on a carbenicillin plate or by an increase in the bacterial β-galactosidase activity. Note: the known interaction between the dimerization domain of yeast transcriptional activator Gal4 (LGF2) and a domain derived from a mutant form of GAL11 (Stratagene) served as a positive control.

Figure 6. Interaction between L0036 and Ler

(A) Interaction of L0036 with Ler shown by co-precipitation in a pull-down experiment. L0036 was fused to the C-terminus of GST, and this recombinant protein was bound to glutathione–Sepharose beads. Subsequent binding of His₆-tagged Ler to the beads was examined by Western blotting using specific antibodies. (B) Co-expressing L0036 (from pCCL36) and Ler (from pACYLerK) decreased the activation effect of Ler on the LEE5 promoter. Bacteria (JM110) carrying a plasmid pKM-LEE5 that has lacZ driven by the LEE5 promoter were transformed by compatible plasmids. The extent of activation of the LEE5 promoter is reflected in the measured bacterial β-galactosidase activity. Measurements were taken from three separate colonies of the transformants.

Figure 7. Fine mapping the region of L0036 involved in the suppression of the LEE expression

(A) Schematic illustration of the truncated L0036 expressed from individual plasmids. His₆-tag fused to the N-terminus of L0036 is represented by hatched bars. (B) Bacterial lysates from the wild-type strain 43888 harbouring additional plasmids shown in (A) were analysed for the LEE proteins by Western blotting as described in legend to Figure 1. Each band on the blot was quantified by ImageQuant version 1.2 (Molecular Dynamics), and the digitalized intensity was compared with that derived solely from the wild-type bacteria. Data were summarized from three different experiments.