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Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea in the developing world (17). EPEC colonizes the small intestine and requires intimate attachment to the host epithelium for full virulence. Intimate attachment leads to the formation of attaching and effacing lesions (A/E lesions), which involve the degeneration of the epithelial brush border, and the formation of actin-rich pedestals within the host cell beneath adherent EPEC (13, 16). A/E lesion formation is dependent on translocation of the bacterial protein Tir (translocated intimin receptor; formerly called Hp90) to the host cell membrane, where it is tyrosine phosphorylated and serves as a receptor for the EPEC outer membrane protein intimin (11). Intimin binding results in the rearrangement of host cytoskeletal proteins and in pedestal formation beneath adherent bacteria. Tir tyrosine phosphorylation is required for EPEC A/E lesion formation (10). Tir translocation and A/E lesion formation are dependent on an intact type III secretory system and the EPEC secreted proteins EspA, EspB, and EspD. Recent work indicates that EspB and EspD are translocated into the host cell (22, 23, 24), while EspA forms filamentous organelles on the EPEC cell surface that are thought to be involved in protein translocation to the host cell (2, 14). The genes for these proteins are located in a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE) (15).

Recently, EPEC strain 30-5-1(3) has been described as deficient in secretion of EspA, EspB, and EspD and lacks the ability to invade cultured epithelial cells (12, 19). EPEC 30-5-1(3) has a TnphoA transposon insertion in sepZ, which is the first open reading frame in a transcriptional unit, LEE2, that contains five additional genes (3, 5, 19). Interestingly, this mutant has also been reported to form A/E lesions without evidence of Tir phosphorylation (Hp90), suggesting that this mutant is deficient in activating host tyrosine kinase-mediated signaling pathways yet is still capable of forming pedestals (19). In view of the recent identification of Hp90 as the bacterial protein Tir and findings suggesting possible functions for EspA, EspB, EspD and the role of type III secretion in Tir delivery, we undertook experiments examining A/E lesion formation by strain 30-5-1(3).

Tir is not secreted by 30-5-1(3). Using enzyme-linked immunosorbent assay (ELISA) techniques, we were unable to detect Tir secretion from EPEC strain 30-5-1(3), whereas under the same conditions, a low level of secreted Tir was observed in wild-type EPEC (Fig. 1). Secreted proteins were prepared from EPEC E2348/69, 30-5-1(3), E2348/69/pCVD450, and 30-5-1(3)/pCVD450 by diluting a standing Luria-Bertani (LB) broth-grown overnight culture 1:100 in Dulbecco's modified Eagle's medium (DMEM) and growing it at 37°C and 5% CO₂ to an optical density at 600 nm (OD₆₀₀) of 0.7 as previously described (11). Tir secretion into the culture supernatant was determined by ELISA using anti-Tir antisera (11). In wild-type EPEC, Tir secretion is enhanced by transformation with pCVD450 (11), which contains a portion of the per regulatory locus (6). We examined the effect of pCVD450 on secretion from EPEC 30-5-1(3) and found that it had no effect, compared to wild-type EPEC (Fig. 1). In agreement with earlier reports (12, 19), we detected no EspB secretion from EPEC 30-5-1(3) (data not shown).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Secretion of Tir by EPEC 30-5-1(3) is not detectable. ELISA wells were coated with secreted proteins from wild-type EPEC (open circles), EPEC transformed with pCVD450 (filled circles), EPEC 30-5-1(3) (open squares), or EPEC 30-5-1(3)/pCVD450 (filled squares) and then incubated with anti-Tir antisera. Each data point represents the mean ± standard deviation of triplicate points from at least three experiments.

30-5-1(3) is attenuated in Tir and EspB translocation and in the formation of EspA filaments. We next examined whether 30-5-1(3) could translocate Tir to the host cell, and if it became tyrosine phosphorylated. HeLa cells were infected with EPEC strains (standing overnight culture; multiplicity of infection [MOI], 1:100) for 2 to 4 h, washed and solubilized with 1% Triton X-100, and microcentrifuged to separate Triton-soluble (membranes and cytosol) and -insoluble (cytoskeleton and adherent bacteria) fractions as described previously (21). Tir translocation and phosphorylation were examined at several time points after infection using Western blotting with rat anti-Tir or anti-PY (4G10; Upstate Biotechnology Inc.) antisera (Fig. 2A). Tir was translocated to the host cell by both wild-type EPEC and strain 30-5-1(3), as evidenced by the appearance of a 90-kDa band recognized by both anti-Tir and anti-PY antisera, in both the Triton X-100-soluble and -insoluble fractions (Fig. 2A, lanes a). In wild-type EPEC, tyrosine-phosphorylated Tir appeared in both the soluble and insoluble fractions as early as 2 h postinfection. There was a considerable delay in the appearance of translocated Tir in cells infected with strain 30-5-1(3). Tyrosine-phosphorylated Tir was detected only after 3 to 4 h postinfection, and its level was significantly lower than that apparent in cells infected by wild-type EPEC (Fig. 2A, lanes b). Tir phosphorylation occurred upon translocation by both strains, resulting in the appearance of a 90-kDa band in an anti-PY Western blot (Fig. 2A, lower panel).

View larger version (37K): [in this window] [in a new window]   FIG. 2.   Reduced translocation of Tir and EspB-CyaA by EPEC 30-5-1(3). (A) Tir is translocated by 30-5-1(3) and tyrosine phosphorylated. HeLa cells were infected with wild-type EPEC or EPEC 30-5-1(3), and Triton X-100-soluble and -insoluble fractions were prepared, and resolved by sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis. After transfer to nitrocellulose membranes, blots were probed with anti-Tir antisera (upper panel) or anti-PY antisera (lower panel). Lanes labeled "a" represent proteins isolated from wild-type EPEC-infected cells, whereas lanes labeled "b" represent proteins from cells infected with 30-5-1(3). Molecular size markers (in kilodaltons) are shown on the left. (B) Translocation of EspB-CyaA by EPEC 30-5-1(3). HeLa cells were infected with EPEC 30-5-1(3)/pEX-EspB-CyaA or wild-type EPEC/pEX-EspB-CyaA, and the percentage of [3H]ATP converted into [3H]cAMP was determined and compared to that in uninfected HeLa cells. Each data point represents the mean ± standard deviation of results from four experiments. Asterisks indicate values significantly different from those for uninfected HeLa cells. Differences between groups were determined using a nonparametric Tukey type multiple comparison test.

View larger version (106K): [in this window] [in a new window]   FIG. 3.   EPEC 30-5-1(3) is attenuated in forming EspA filaments. DMEM-grown bacteria were stained with rabbit anti-EspA followed by an anti-rabbit antibody conjugated with FITC. (A and B) Immunofluorescence and corresponding phase image for wild-type EPEC, respectively; (C and D) immunofluorescence and corresponding phase image for EPEC 30-5-1(3), respectively.

EPEC 30-5-1(3) forms wild-type A/E lesions but is noninvasive. HeLa cells were infected with either wild-type EPEC or EPEC 30-5-1(3), and at 2 or 4 h postinfection, cells were prepared for immunofluorescence microscopy by paraformaldehyde fixation and labeled with either anti-PY antisera or rat anti-Tir, and Texas red phalloidin. To quantify adherence and pedestal formation, three replicate fields of 100 cells were assessed for bacterial adherence and A/E lesion formation by anti-PY and phalloidin labeling. Adherence was calculated as the percentage of cells with microcolonies of 5 or more bacteria, whereas A/E lesion formation was calculated as the percentage of infected cells showing tightly focused actin pedestals beneath adhering EPEC. The differences between groups were determined by analysis of variance (ANOVA). After a 2-h infection, wild-type EPEC formed small microcolonies, with actin and tyrosine-phosphorylated proteins evident under most adhering bacteria (Fig. 4A). This correlated with the appearance of tyrosine-phosphorylated Tir as shown by Western analysis (Fig. 2A). In contrast, although EPEC 30-5-1(3) formed small microcolonies after 2 h, there was little actin or PY beneath adhering bacteria. Although there was no significant difference in the level of adherence between the two strains [49.8 ± 17.2% for the wild type versus 35.5 ± 5.3% for 30-5-1(3); P = 0.18; n = 3], EPEC 30-5-1(3) infection resulted in the formation of significantly fewer A/E lesions [53.0 ± 15.1% for the wild type versus 8.9 ± 4.8% for 30-5-1(3); P = 0.01; n = 3]. When examined after 4 h, EPEC 30-5-1(3) induced the formation of actin-rich pedestals, with tyrosine-phosphorylated proteins located at the tip, in a manner indistinguishable from wild-type EPEC (Fig. 4B and C). At this time point, there was no significant difference in the level of adherence [for the wild type, 99.4 ± 1.1%; for 30-5-1(3), 99.7 ± 0.6%; P = 0.2; n = 3] or A/E lesion formation [for the wild type, 96.1 ± 3.2%; for 30-5-1(3), 97.6 ± 3.3%; P = 0.47; n = 3] between the two strains. Although EPEC 30-5-1(3) was able to form A/E lesions, it was significantly attenuated in its ability to invade HeLa cells. We observed a 50- to 100-fold decrease in invasion with no effect on bacterial adherence upon infection with EPEC 30-5-1(3) compared to infection with wild-type EPEC (data not shown). These data are identical to those reported by Rabinowitz and colleagues (19).

View larger version (76K): [in this window] [in a new window]   FIG. 4.   EPEC 30-5-1(3) forms wild-type A/E lesions. (A and B) Immunofluorescence microscopy of HeLa cells infected with EPEC 30-5-1(3) or wild-type EPEC and fixed, permeabilized, and colabeled with anti-PY (FITC) and Texas red phalloidin after 2 (A) or 4 (B) h of infection. (C) HeLa cells infected with either EPEC 30-5-1(3) or wild-type EPEC for 4 h, and labeled with anti-PY (FITC) and anti-Tir (rhodamine).