Microarray Analysis of the Ler Regulon in Enteropathogenic and Enterohaemorrhagic Escherichia coli Strains

Lewis E H Bingle,Stephen J W Busby,Chrystala Constantinidou,David J Lee,Charles W Penn,Mala Patel,Md Shahidul Islam,Mark J Pallen,Robert K Shaw,Lori A S Snyder,David A Rasko

doi:10.1371/journal.pone.0080160

Abstract

The type III protein secretion system is an important pathogenicity factor of enteropathogenic and enterohaemorrhagic Escherichia coli pathotypes. The genes encoding this apparatus are located on a pathogenicity island (the locus of enterocyte effacement) and are transcriptionally activated by the master regulator Ler. In each pathotype Ler is also known to regulate genes located elsewhere on the chromosome, but the full extent of the Ler regulon is unclear, especially for enteropathogenic E. coli. The Ler regulon was defined for two strains of E. coli: E2348/69 (enteropathogenic) and EDL933 (enterohaemorrhagic) in mid and late log phases of growth by DNA microarray analysis of the transcriptomes of wild-type and ler mutant versions of each strain. In both strains the Ler regulon is focused on the locus of enterocyte effacement – all major transcriptional units of which are activated by Ler, with the sole exception of the LEE1 operon during mid-log phase growth in E2348/69. However, the Ler regulon does extend more widely and also includes unlinked pathogenicity genes: in E2348/69 more than 50 genes outside of this locus were regulated, including a number of known or potential pathogenicity determinants; in EDL933 only 4 extra-LEE genes, again including known pathogenicity factors, were activated. In E2348/69, where the Ler regulon is clearly growth phase dependent, a number of genes including the plasmid-encoded regulator operon perABC, were found to be negatively regulated by Ler. Negative regulation by Ler of PerC, itself a positive regulator of the ler promoter, suggests a negative feedback loop involving these proteins.

Highlights

Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli are two pathotypes of this important gastrointestinal bacterium that can cause serious diarrhoeal disease in humans [1]
Many EHEC and encoded regulator PerC (EPEC) strains possess a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE) that is found in the related bacterium Citrobacter rodentium, a mouse pathogen that is widely used as a model for the EHEC and EPEC strains [2]
We constructed two validated mutant strains of E. coli: LBEC1 (EDL933 Dler) and LBEC2 (E2348/69 Dler), grew cultures of WT and parental mutant strains under conditions known to be inducing for the LEE to two different growth phases, harvested RNA and used this to perform microarray analysis of the transcriptomes

Summary

Introduction

Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli are two pathotypes of this important gastrointestinal bacterium that can cause serious diarrhoeal disease in humans [1]. Many EHEC and EPEC strains possess a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE) that is found in the related bacterium Citrobacter rodentium, a mouse pathogen that is widely used as a model for the EHEC and EPEC strains [2]. There are significant differences in overall pathogenicity between EHEC and EPEC strains, for example EHEC strains cause a more severe bloody diarrheal disease (haemorrhagic colitis) that is often accompanied by the life threatening complication, haemolytic uraemic syndrome (HUS) [8]. Such differences are presumably mainly determined by the differing contributions of the extra-LEE factors. Examples include differing arrays of T3SS effector proteins and the fact that the EHEC genome encodes a Shiga-like toxin responsible for serious pathology in the human host, while EPEC does not [8]

Methods

Results

Conclusion