Atypical enteropathogenic Escherichia coli that contains functional locus of enterocyte effacement genes can be attaching-and-effacing negative in cultured epithelial cells

Abstract

Enteropathogenic Escherichia coli (EPEC) induces a characteristic histopathology on enterocytes known as the attaching-and-effacing (A/E) lesion, which is triggered by proteins encoded by the locus of enterocyte effacement (LEE). EPEC is currently classified as typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence or absence of the EPEC adherence factor plasmid, respectively. Here we analyzed the LEE regions of three aEPEC strains displaying the localized adherence-like (LAL), aggregative adherence (AA), and diffuse adherence (DA) patterns on HEp-2 cells as well as one nonadherent (NA) strain. The adherence characteristics and the ability to induce A/E lesions were investigated with HeLa, Caco-2, T84, and HT29 cells. The adherence patterns and fluorescent actin staining (FAS) assay results were reproducible with all cell lines. The LEE region was structurally intact and functional in all strains regardless of their inability to cause A/E lesions. An EspF(U)-expressing plasmid (pKC471) was introduced into all strains, demonstrating no influence of this protein on either the adherence patterns or the capacity to cause A/E of the adherent strains. However, the NA strain harboring pKC471 expressed the LAL pattern and was able to induce A/E lesions on HeLa cells. Our data indicate that FAS-negative aEPEC strains are potentially able to induce A/E in vivo, emphasizing the concern about this test for the determination of aEPEC virulence. Also, the presence of EspF(U) was sufficient to provide an adherent phenotype for a nonadherent aEPEC strain via the direct or indirect activation of the LEE4 and LEE5 operons.

Fig. 1.

Adherence assay and FAS test of wild-type aEPEC and aEPEC strains harboring pKC471 performed in 6 h using HeLa cells. Typical EPEC strain E2348/69 and E. coli strain DH5α were used as positive and negative adherence controls, respectively.

Fig. 2.

Transcriptional profile of LEE operons of aEPEC strains. In experiments performed with DMEM and HeLa cells, transcriptional levels of ler (LEE1) (A and B, respectively), escC (LEE2) (C and D, respectively), escV (LEE3) (E and F, respectively), espA (LEE4) (G and H, respectively), and eae (LEE5) (I and J, respectively) were measured by real-time PCR. Relative fold expression represents the change (n-fold) in the transcriptional level compared to the level of LAL strain BA320 (gray bar, value of 1.0). Results are expressed as means and standard deviations of data from triplicate experiments. The levels of the rpoA transcript were used to normalize the C_T values to account for variations in bacterial numbers. Statistical significance was determined by a Student's t test based on comparisons with strain BA320 (*, P < 0.05; ⊠, P ≥ 0.05 [not significant]).

Fig. 3.

Expression of intimin, Tir, EspA, EspB, and EspD of aEPEC strains analyzed by immunoblotting. Typical EPEC strain E2348/69 was used as a positive control, and the following mutants were used as negative controls: CVD206 (eae mutant), EPEC Δtir (tir), UMD872 (espA mutant), UMD864 (espB mutant), and UMD870 (espD mutant). The whole-cell proteins intimin and Tir were separated on 12% SDS-PAGE gels. The secreted proteins EspA, EspB, and EspD were separated on 10% SDS-PAGE gels. The apparent molecular masses are indicated on the left.