Comparative analysis of locus of enterocyte effacement pathogenicity islands of atypical enteropathogenic Escherichia coli

Abstract

The pathogenicity of enteropathogenic Escherichia coli (EPEC) is linked to the locus of enterocyte effacement, or LEE, encoding a type III secretion system (T3SS) that directly transfers bacterial effector proteins into eukaryotic cells. Atypical diffusely adhering EPEC (DA-EPEC) strains that harbor homologues of the LEE but lack the EPEC adherence factor plasmid have been increasingly associated with outbreaks of diarrhea. In this study, we have completely sequenced and functionally characterized LEE pathogenicity islands derived from the clinical DA-EPEC isolates 3431 (O8:H-) and 0181 (O119:H9:K61). LEE3431 and LEE0181 exhibit genetic organization analogous to that of the prototype LEE(E2348/69). Genes constituting the T3SS apparatus are highly conserved. However, LEE-encoded effector proteins exhibit major differences. Transfer and functional expression of LEE0181 in an E. coli XL1 blue MR background demonstrated that LEE0181 contains all the information for signal transduction and pedestal formation.

FIG. 1.

Organization of the prototype LEE_E2348/69 and analogous segments of atypical LEEs covered by cosmid clones. The cosmid clone LEE₀₁₈₁ covers the complete LEE of the DA-EPEC strain 0181, and the cosmid LEE₃₄₃₁ encompasses 28,251 bp of the LEE of the DA-EPEC strain 3431. The sequence between the eae and the espD genes of LEE₃₄₃₁ was obtained using a 3-kb PCR fragment; the 3′ sequence of LEE₃₄₃₁ from the espD gene to the 3′ end had been determined previously.

FIG. 2.

Schematic overview of the chromosomal junctions of the LEEs of the DA-EPEC strains 0181 and 3431.

FIG. 3.

Alignments of the EspF protein of the EPEC protoype strain E2348/69 with those of the DA-EPEC strains 0181 and 3431. Residues differing from those of EspF_E2348/69 are boxed in black. The three proline-rich repeats in EspF are shaded in grey.

FIG. 4.

Phylogenetic tree of intimin proteins of DA-EPEC strains, the prototype EPEC strain E2348/69, the EHEC strain EDL933, and the rabbit EPEC strain RDEC-1 as a visualization of sequence alignments. The tree was generated using DNASTAR MegAlign software.

FIG. 5.

Transmission electron microscopy of a HeLa cell infected with JG-LEE₀₁₈₁/XL1 blue MR (magnification, ×19,000). The recombinant strain is able to adhere to HeLa cells and induces the formation of host cell protrusions (pedestals) underneath adherent bacteria.