The locus of enterocyte effacement-encoded effector proteins all promote enterohemorrhagic Escherichia coli pathogenicity in infant rabbits

Abstract

The genes encoding the enterohemorrhagic Escherichia coli (EHEC) type III secretion system (TTSS) and five effector proteins secreted by the TTSS are located on the locus of enterocyte effacement (LEE) pathogenicity island. Deletion of tir, which encodes one of these effector proteins, results in a profound reduction (approximately 10,000-fold) in EHEC colonization of the infant rabbit intestine, but the in vivo phenotypes of other LEE genes are unknown. Here, we constructed in-frame deletions in escN, the putative ATPase component of the TTSS, and the genes encoding the four other LEE-encoded effector proteins, EspH, Map, EspF, and EspG, to investigate the contributions of the TTSS and the translocated effector proteins to EHEC pathogenicity in infant rabbits. We found that the TTSS is required for EHEC colonization and attaching and effacing (A/E) lesion formation in the rabbit intestine. Deletion of escN reduced EHEC recovery from the rabbit intestine by approximately 10,000-fold. Although EspH, Map, EspF, and EspG were not required for A/E lesion formation in the rabbit intestine or in HeLa cells, these effector proteins promote EHEC colonization. Colonization by the espH and espF mutants was reduced throughout the intestine. In contrast, colonization by the map and espG mutants was reduced only in the small intestine, indicating that Map and EspG have organ-specific effects. EspF appears to down-regulate the host response to EHEC, since we observed increased accumulation of polymorphonuclear leukocytes in the colonic mucosa of rabbits infected with the EHEC espF mutant. Thus, all the known LEE-encoded effector proteins influence EHEC pathogenicity.

FIG. 1.

LEE pathogenicity island of E. coli O157:H7 strain EDL933. The open reading frames encoding the translocated effector proteins are shown as light grey arrows, and escN is depicted as a white arrow. This figure incorporates information reported in references , , , , and . The putative polycistronic operons of EHEC LEE are shown as thin dotted lines.

FIG. 2.

Transmission electron micrographs of colonic tissue taken from rabbits infected with EHEC 905ΔescN (A), 905 (B), 905ΔespH (C), 905Δmap (D), 905ΔespG (E), and 905ΔespF (F). Colon samples were taken 2 days postinoculation.

FIG. 3.

Recovery of 905 or one of its derivatives from intestinal segments or stool samples from infected rabbits 7 days postinoculation. Samples below the detection limit (open squares) and scores for rabbits inoculated with the independently derived espH or espF deletion mutants (×) are indicated. The mean value for each strain is indicated by a bar.

FIG. 4.

Colitis scores from the midcolons of infant rabbits inoculated with 905 or one of its derivatives. Sections stained with hematoxylin and eosin were scored for heterophils (A), edema and congestion (B), and mucosal damage (C) as described previously (44, 52). The scores for rabbits inoculated with the independently derived espF deletion mutant (×) are indicated. The median value for each strain is indicated by a bar.