Development of a universal intimin antiserum and PCR primers

Abstract

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) constitute a significant risk to human health worldwide. A hallmark of both pathogens is their ability to produce characteristic attaching-and-effacing (A/E) lesions in intestinal epithelial cells. Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Intimin, an LEE-encoded bacterial adhesion molecule, mediates the intimate bacterium-host cell interaction characteristic of A/E lesions. On the basis of characterization of the C-terminal 280-amino-acid cell binding domain of intimin (Int280(661-939)), four distinct Int280 types (types alpha, beta, gamma, and delta) have been identified. Importantly, Int280alpha and Int280beta antisera specifically recognized their respective intimin types. Using a conserved region of the intimin molecule (Int(388-667)) and primers synthesized to generate the recombinant Int(388-667), we have now generated universal intimin antiserum and PCR primers that are reactive with the different intimin types expressed by both human and animal A/E lesion-forming strains. Use of immunogold electron microscopy to visualize intimin on the surfaces of EPEC and EHEC strains revealed, in general, a uniform distribution on the bacterial cell surface. However, a filamentous staining pattern was observed with a few strains expressing intimin gamma. Cloning of the intimin eae gene from one such strain (strain ICC57) into strain CVD206, an EPEC strain which harbors a null deletion in eae, produced a uniform intimin staining pattern indicating that, if the filamentous staining pattern defines a filamentous form of intimin gamma, it is dependent upon the genetic background of the strain and is not a feature of the intimin molecule.

FIG. 1

Western blot analysis of Int_388–667 antiserum against various A/E lesion-forming E. coli strains. (a) The antiserum detected intimin α from strain E2348/69 (lane 1), while no reactivity was observed with eae-negative strain CVD206, which was used as a negative control (lane 2). (b) Similar levels of intimin expression are shown by representative human isolates: O111:H2 (intimin β; lane 1), O119:H6 (intimin β; lane 2), O55:H7 (intimin γ; lane 3), O55:H− (intimin γ; lane 4), and O157:H7 (intimin γ; lane 5).

FIG. 2

Immunogold labelling of intimin. The specificity of the Int_388–667 antibody was confirmed by labelling wild-type EPEC strain E2348/69 (a) and its intimin-negative derivative, CVD206 (b). The antiserum labelled all strains that express intimins α, β, and δ, although there was some strain-to-strain variation in the level of intimin expression illustrated here, with two strains expressing intimins α (c) and β (d), respectively. Magnification, ×30,000.

FIG. 3

Detection of different intimin types with the universal intimin primers. A specific PCR product (840 bp) was generated from a human O127 EPEC isolate expressing intimin α (lane 2), cow O26 isolates expressing intimin β (lanes 4 and 8), sheep O157:H7 and O157:H− isolates expressing intimin γ (lanes 5 and 6, respectively), a cow O157:H7 isolate expressing intimin γ (lane 7), and a pig O26 isolate expressing intimin β (lane 10). No PCR product was obtained with CVD206 (lane 3), a cow O26 isolate expressing intimin β (lane 9), L. monocytogenes (lane 11), or Y. pseudotuberculosis (lane 12). Molecular size markers (1-kb ladder) were loaded in lane 1.

FIG. 4

Immunogold labelling of intimin γ The antiserum labelled all strains expressing intimin γ, and most strains including EHEC 85-170 (O157:H7) (a) displayed a uniform distribution of surface intimin, although strains ICC39 (O55:H−) and ICC57 (O55:H7) (b) showed a filamentous pattern of intimin staining. Intimin γ from strain ICC57 cloned into CVD206 [strain CVD206(pICC57)] displayed a uniform surface intimin distribution (c). Magnification, ×30,000.

FIG. 5

Corresponding actin fluorescence (a and c) and phase-contrast micrographs (b and d) showing HEp-2 cells infected with EPEC ICC57 (a and b) and CVD206(pICC57) (c and d) for 3 h. Both strains produced intense spots of actin fluorescence at sites of bacterial attachment (positive FAS test) indicative of A/E lesion formation. However, strain CVD206(pICC57) was atypical in that it produced a stellate distribution of actin accretion (c, arrow).