The Two-Component System CpxRA Negatively Regulates the Locus of Enterocyte Effacement of Enterohemorrhagic Escherichia coli Involving σ(32) and Lon protease.

Miguel A De La Cruz,Jason K Morgan,James T Riordan,Jorge A Girón,Jorge A Yáñez-Santos,Miguel A Ares

doi:10.3389/fcimb.2016.00011

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a significant cause of serious human gastrointestinal disease worldwide. EHEC strains contain a pathogenicity island called the locus of enterocyte effacement (LEE), which encodes virulence factors responsible for damaging the gut mucosa. The Cpx envelope stress response of E. coli is controlled by a two-component system (TCS) consisting of a sensor histidine kinase (CpxA) and a cytoplasmic response regulator (CpxR). In this study, we investigated the role of CpxRA in the expression of LEE-encoded virulence factors of EHEC. We found that a mutation in cpxA significantly affected adherence of EHEC to human epithelial cells. Analysis of this mutant revealed the presence of high levels of CpxR which repressed transcription of grlA and ler, the main positive virulence regulators of the LEE, and influenced negatively the production of the type 3 secretion system–associated EspABD translocator proteins. It is known that CpxR activates rpoH (Sigma factor 32), which in turns activates transcription of the lon protease gene. We found that transcription levels of ler and grlA were significantly increased in the lon and cpxA lon mutants suggesting that lon is involved in down-regulating LEE genes. In addition, the Galleria mellonella model of infection was used to analyze the effect of the loss of the cpx and lon genes in EHEC's ability to kill the larvae. We found that the cpxA mutant was significantly deficient at killing the larvae however, the cpxA lon mutant which overexpresses LEE genes in vitro, was unable to kill the larvae, suggesting that virulence in the G. mellonella model is T3SS independent and that CpxA modulates virulence through a yet unknown EHEC-specific factor. Our data provides new insights and broadens our scope into the complex regulatory network of the LEE in which the CpxA sensor kinase plays an important role in a cascade involving both global and virulence regulators.

Highlights

Human infections with enterohemorragic Escherichia coli (EHEC) O157:H7 can cause diarrhea ranging from mild to bloody and can induce hemorrhagic colitis (Riley et al, 1983; Blattner et al, 1997; Jonson et al, 2005)
Most of the gene products required for the formation of AE lesions by EHEC are present in a pathogenicity island called the locus of enterocyte effacement (LEE)
We propose a model of how this two-component system (TCS) interplays with other known and unknown regulatory networks to modulate expression of virulence factors encoded in the LEE pathogenicity island

Summary

Introduction

Human infections with enterohemorragic Escherichia coli (EHEC) O157:H7 can cause diarrhea ranging from mild to bloody and can induce hemorrhagic colitis (Riley et al, 1983; Blattner et al, 1997; Jonson et al, 2005). Hallmarks of EHEC pathogenicity are their ability to produce one or two Shiga toxins (Stx and Stx2), which are responsible for the HUS (Karmali et al, 1983; Paton and Paton, 1998) and colonization of the gut mucosa leading to the development of intestinal attaching and effacing (AE) lesions (Nataro and Kaper, 1998). Most of the gene products required for the formation of AE lesions by EHEC are present in a pathogenicity island called the locus of enterocyte effacement (LEE). The regulation of the LEE of EHEC involves a complex regulatory network comprising virulence (Ler, GrlA, and GrlR) and global regulators such as Pch, H-NS, IHF, Hha, Fis, BipA, QseA, GrvA, EtrA, YhiEF, SdiA, among others (Mellies et al, 2007)

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