Attaching and effacing (A/E) lesion formation by enteropathogenic E. coli on human intestinal mucosa is dependent on non-LEE effectors.

Massiel Cepeda-Molero,Gad Frankel,Stephanie Schüller,Alistair D S Walsham,Cedric N Berger,Samuel J Ellis,Simon Wemyss-Holden,Luis Ángel Fernández,Brian K Coombes

doi:10.1371/journal.ppat.1006706

Abstract

Enteropathogenic E. coli (EPEC) is a human pathogen that causes acute and chronic pediatric diarrhea. The hallmark of EPEC infection is the formation of attaching and effacing (A/E) lesions in the intestinal epithelium. Formation of A/E lesions is mediated by genes located on the pathogenicity island locus of enterocyte effacement (LEE), which encode the adhesin intimin, a type III secretion system (T3SS) and six effectors, including the essential translocated intimin receptor (Tir). Seventeen additional effectors are encoded by genes located outside the LEE, in insertion elements and prophages. Here, using a stepwise approach, we generated an EPEC mutant lacking the entire effector genes (EPEC0) and intermediate mutants. We show that EPEC0 contains a functional T3SS. An EPEC mutant expressing intimin but lacking all the LEE effectors but Tir (EPEC1) was able to trigger robust actin polymerization in HeLa cells and mucin-producing intestinal LS174T cells. However, EPEC1 was unable to form A/E lesions on human intestinal in vitro organ cultures (IVOC). Screening the intermediate mutants for genes involved in A/E lesion formation on IVOC revealed that strains lacking non-LEE effector/s have a marginal ability to form A/E lesions. Furthermore, we found that Efa1/LifA proteins are important for A/E lesion formation efficiency in EPEC strains lacking multiple effectors. Taken together, these results demonstrate the intricate relationships between T3SS effectors and the essential role non-LEE effectors play in A/E lesion formation on mucosal surfaces.

Highlights

The gastrointestinal epithelium is an important defense barrier against infections [1]
By generating deletion mutants lacking combinations or the whole repertoire of protein effectors encoded by Enteropathogenic E. coli (EPEC), we show that intimin-translocated intimin receptor (Tir) interaction is not sufficient and reveal an additive role of non-locus of enterocyte effacement (LEE) effectors for A/E lesion formation in human intestinal tissue
We employed a marker-less deletion/replacement strategy to generate a library of EPEC effector mutants, which allow multiple deletions and/or integrations while leaving neither an antibiotic gene cassette nor short heterologous DNA sequences ("scars") in the chromosome [34]

Summary

Introduction

The gastrointestinal epithelium is an important defense barrier against infections [1]. Enteric pathogens have acquired virulence traits that enable them to colonize and break this barrier, by adhering to the epithelium, delivering toxins and invading intestinal epithelial cells. To this end, several important human and animal pathogens employ type III secretion systems (T3SS) to inject virulence factors into infected eukaryotic cells, where they take control of cell signaling [2]. In HeLa cells, clustering of intimin with its receptor Tir [8] triggers robust actin polymerization leading to formation of pedestal-like structures [4, 9]. Intimin–Tir interaction is necessary for A/E lesion formation, but it is not currently known if this binding is sufficient [10]

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