The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli

Abstract

Regulation of virulence gene expression in enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is incompletely understood. In EPEC, the plasmid-encoded regulator Per is required for maximal expression of proteins encoded on the locus of enterocyte effacement (LEE), and a LEE-encoded regulator (Ler) is part of the Per-mediated regulatory cascade upregulating the LEE2, LEE3, and LEE4 promoters. We now report that Ler is essential for the expression of multiple LEE-located genes in both EPEC and EHEC, including those encoding the type III secretion pathway, the secreted Esp proteins, Tir, and intimin. Ler is therefore central to the process of attaching and effacing (AE) lesion formation. Ler also regulates the expression of LEE-located genes not required for AE-lesion formation, including rorf2, orf10, rorf10, orf19, and espF, indicating that Ler regulates additional virulence properties. In addition, Ler regulates the expression of proteins encoded outside the LEE that are not essential for AE lesion formation, including TagA in EHEC and EspC in EPEC. delta ler mutants of both EPEC and EHEC show altered adherence to epithelial cells and express novel fimbriae. Ler is therefore a global regulator of virulence gene expression in EPEC and EHEC.

FIG. 1

The LEE, showing the structures of the LEE1 through LEE4 and tir operons and the mapped promoters. The promoter driving ler expression (P_LEE1) has been expanded to show the differences between the promoter of EPEC O127:H6 and those of EHEC O157:H7 and EPEC O55:H7. EPEC contains an ATAAGG duplication that disrupts the region corresponding to the −10 sequence found in EHEC (33, 42). Open reading frames have been shaded to distinguish those encoding secreted proteins from those involved in type III secretion or other functions. In the alignment of the sequences, /…/ represents an area deleted from the figure for presentation and − represents a nucleotide missing in that sequence when compared with the other sequence.

FIG. 2

Comparison of Ler and its homologs. Ler homologs are aligned to show identical (marked by their letter) and similar but nonidentical (+) amino acids. Ler from EHEC and EPEC are highly conserved and are related to the H-NS family of DNA binding proteins, including BbH3 and BpH3 of Bordetella, StpA, other H-NS orthologs and paralogs, and, more distantly, E. coli H-NS. Similarity is highest at the C-terminal region, which mediates DNA binding. The conserved DNA binding motif (TWTGXGRXP) contained in Ler and all members of the H-NS family is underlined (6). The percent identity (%ID) and similarity (%Sm) over the homologous region are listed to the right of each alignment, as is the number of gap initiations in the alignment (#gaps, gaps not shown).

FIG. 3

Mutation of ler affects the production of LEE-encoded proteins. Western blots of secreted proteins probed with antibodies against all-EPEC secreted proteins (A) or Tir (B), membrane preparations stained with antibodies against the outer membrane protein intimin (C), and whole-cell lysates stained with antisera against EspF (D) or rOrf2 (E) indicate that multiple LEE-encoded virulence factors are regulated by Ler. wt, wild type.

FIG. 4

Regulation of high-molecular-mass secreted proteins by Ler. (A) Coomassie blue-stained gels of secreted protein preparations from EPEC and EHEC demonstrated that ∼110-kDa proteins were not secreted from Δler mutants. (B) Antibodies identify this protein in EPEC supernatants as EspC. wt, wild type.

FIG. 5

Altered adherence phenotypes of Δler mutants. (A to C) EPEC (A) and the complemented Δler mutant (C) exhibited LA and formed large microcolonies on the surface of the HEp-2 cells; the EPEC Δler mutant (B) displayed a complex mixture of DA and AA patterns with some LA microcolony formation. (D to F) With EHEC 85-170, the wild type adhered to HEp-2 cells in a DA-LA pattern (D) while the Δler mutant displayed increased adherence, especially to glass, and an AA pattern (E); complementation of the Δler mutation with plasmid-encoded ler restored the wild-type adherence phenotype (F). Magnification, ×2,500.

FIG. 6

Novel fimbriae expressed by Δler mutants. (A and B) The EPEC Δler mutant expressed a number of novel fimbriae (A, a and c) that are clearly distinguishable from BFP (b) and include numerous LFF (c) as well as “bent” fimbriae (b) and shorter fine fimbriae (B, arrow). (C) EHEC 85-170 does not express fimbriae (not shown), but the Δler mutant exhibited long fine fimbriae (arrow). Bar = 200 μm.

FIG. 7

Regulation of multiple EPEC and EHEC genes by Ler. Primer extension was performed on mRNA extracted from Δler mutants (−) and Ler⁺ complements (+) of EHEC (∗) or EPEC (no ∗) by using primers K848 through K1967, which are directed against transcripts from genes rorf2 through espP, as shown. The resultant cDNA transcripts were visualized by autoradiography.

FIG. 8

The ler regulon. Ler regulates the expression of many genes both within the LEE and elsewhere on the genome. Some genes are directly regulated by Ler, as shown in gene fusion studies in E. coli K-12 (indicated by solid lines), while other genes are indirectly regulated or direct regulation has not been demonstrated (dashed lines). Expression of Ler is activated by quorum sensing in both EPEC and EHEC and additionally by Per in EPEC. Genes in the top half of the figure apply to EPEC O127:H6, and genes in the bottom half apply to EHEC O157:H7.