Complete nucleotide sequence and analysis of the locus of enterocyte Effacement from rabbit diarrheagenic Escherichia coli RDEC-1

Abstract

The pathogenicity island termed the locus of enterocyte effacement (LEE) is found in diverse attaching and effacing pathogens associated with diarrhea in humans and other animal species. To explore the relation of variation in LEE sequences to host specificity and genetic lineage, we determined the nucleotide sequence of the LEE region from a rabbit diarrheagenic Escherichia coli strain RDEC-1 (O15:H-) and compared it with those from human enteropathogenic E. coli (EPEC, O127:H6) and enterohemorrhagic E. coli (EHEC, O157:H7) strains. Differing from EPEC and EHEC LEEs, the RDEC-1 LEE is not inserted at selC and is flanked by an IS2 element and the lifA toxin gene. The RDEC-1 LEE contains a core region of 40 open reading frames, all of which are shared with the LEE of EPEC and EHEC. orf3 and the ERIC (enteric repetitive intergenic consensus) sequence present in the LEEs of EHEC and EPEC are absent from the RDEC-1 LEE. The predicted promoters of LEE1, LEE2, LEE3, tir, and LEE4 operons are highly conserved among the LEEs, although the upstream regions varied considerably for tir and the crucial LEE1 promoter, suggesting differences in regulation. Among the shared genes, high homology (>95% identity) between the RDEC-1 and the EPEC and EHEC LEEs at the predicted amino acid level was observed for the components of the type III secretion apparatus, the Ces chaperones, and the Ler regulator. In contrast, more divergence (66 to 88% identity) was observed in genes encoding proteins involved in host interaction, such as intimin (Eae) and the secreted proteins (Tir and Esps). A comparison of the highly variable genes from RDEC-1 with those from a number of attaching and effacing pathogens infecting different species and of different evolutionary lineages was performed. Although RDEC-1 diverges from some human-infecting EPEC and EHEC, most of the variation observed appeared to be due to evolutionary lineage rather than host specificity. Therefore, much of the observed hypervariability in genes involved in pathogenesis may not represent specific adaptation to different host species.

FIG. 1

(A) Physical map of the RDEC-1 LEE. The orientation of each individual gene is indicated by the direction of the arrow. (B) Amino acid identity of conserved genes between RDEC-1 and E2348/69 (GenBank accession no. AF022236) and between RDEC-1 and EDL933 (GenBank accession no. AF071034). Arrows under the bars indicate the major polycistronic operons corresponding to those in panel A.

FIG. 2

Schematic representation of homologous and unique sequences in the LEE of RDEC-1, E2348/69, EDL933, and the lifA gene of E2348/69 (GenBank accession no. AJ133705). Homologous sequences are indicated by boxes with the same pattern. The dashed box indicates the 34-kb conserved core region of LEE. The flanking regions of the LEE are divergent among RDEC-1, E2348/69, and EDL933.

FIG. 3

(A) Alignment of EspF of EHEC (O157:H7, strain EDL933), EPEC (O126:H7, strain E2348/69), REPEC O103:H2 (strain B10, GenBank accession no. AF116900), and RDEC-1, showing repeated regions (repeats 1, 2, 3, and 4). A 31- to 35-aa conserved region is indicated in bold. Identical nucleotides are indicated by dots. Dashes indicate absent nucleotides. Numbers on the right side of the sequence indicate the amino acid number. (B) Comparison of EspF of RDEC-1 and B10. Identical amino acids are indicated by shaded bars. Numbers above the box indicate the positions of the amino acids. EspF of RDEC-1 is 47 aa (position indicated by the hatched box) shorter than EspF of B10. The sequence of the conserved 35-aa region is indicated in bold.