Intimin type influences the site of human intestinal mucosal colonisation by enterohaemorrhagic Escherichia coli O157:H7

Abstract

Background: Enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli epithelial cell adhesion is characterised by intimate attachment, and attaching and effacing (A/E) lesion formation. This event is mediated in part by intimin binding to another bacterial protein, Tir (translocated intimin receptor), which is exported by the bacteria and integrated into the host cell plasma membrane. Importantly, EPEC (O127:H6) and EHEC (O157:H7) express antigenically distinct intimin types known as intimin alpha and gamma, respectively. EHEC (O157:H7) colonises human intestinal explants although adhesion is restricted to the follicle associated epithelium of Peyer's patches. This phenotype is also observed with EPEC O127:H6 engineered to express EHEC intimin gamma.

Aims: To investigate the influence of intimin on colonisation of human intestine by E coli O157:H7, and intimin types on tissue tropism in humans.

Methods: Human intestinal in vitro organ culture with wild type and mutant strains of O157:H7 were employed.

Results: Introducing a deletion mutation in the eae gene encoding intimin gamma in EHEC (O157:H7) caused the strain (ICC170) to fail to colonise human intestinal explants. However, colonisation of Peyer's patches and A/E lesion formation were restored with intimin gamma expression from a plasmid (ICC170 (pICC55)). In contrast, complementing the mutation with intimin alpha resulted in a strain (ICC170 (pCVD438)) capable of colonising and producing A/E lesions on both Peyer's patch and other small intestinal explants.

Conclusion: Intimin is necessary for human intestinal mucosal colonisation by E coli O157:H7. Intimin type influences the site of colonisation in a Tir type independent mechanism; intimin gamma appears to restrict colonisation to human follicle associated epithelium.

Figure 1

Schematic representation showing construction of the eae deletion of enterohaemorrhagic Escherichia coli (EHEC) strain 85-170. NruI endonuclease digestion of plasmid pCVD444 was used to introduce deletion in the eae gene encoding intimin γ. Following re-ligation and polymerase chain reaction amplification, the DNA fragment containing the deleted form of the eae gene was cloned in the suicide vector pCVD442 and the deletion was introduced into EHEC by allelic exchange, as described in experimental procedures. The numbers represent nucleotides within the structural eae gene. The genes upstream (3` end of tir and cesT) and downstream (escD) of eae are also indicated. (Not to scale.)

Figure 2

Western blot analysis of whole cell lysates prepared from enterohaemorrhagic Escherichia coli (EHEC) 85-170 and the ICC170 derivatives using a universal broad spectrum polyclonal intimin antiserum. Lysates from the wild type (lane 1) and from ICC170 (pCVD438) (lane 3) strains reacted with the intimin antiserum, but not from ICC170 (lane 2).

Figure 3

(A) Lymphoid follicles within Peyer's patch of distal ileum (arrow); bar=100 μm. (B) ICC170 (pICC55) adhering to follicle associated epithelium (FAE) and showing attaching and effacing (A/E) lesion formation; bar=5 μm. (C–E) ICC170 (pCVD438) A/E lesion formation on the FAE of the Peyer's patch, duodenum, and ileum, respectively; bar=5 μm, 1 μm, and 1 μm, respectively. (F) ICC170 (pCVD438) adhesion to the duodenum without A/E lesion formation; bar=1 μm. (G) Lack of adhesion of ICC170 (pCVD438/01) to the ileum; bar=10 μm.