Generation of <i>Escherichia coli</i> intimin derivatives with differing biological activities using site‐directed mutagenesis of the intimin C‐terminus domain

Frankel, Gad; Philips, Alan D.; Nováková, Michaela; Batchelor, Miranda; Hicks, Susan; Dougan, Gordon

doi:10.1046/j.1365-2958.1998.00950.x

Cited by 57 publications

(50 citation statements)

References 43 publications

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“…1. Two unique restriction endonuclease sites located in pCVD438 were used, a conserved Sal I site located upstream of the Int280 domain (position 1663 of the eae gene) and an Eag I site located downstream of the TAA stop codon and within the pACYC184 vector plasmid (Frankel et al , 1998). The DNA fragment between the Sal I site and the 3′ end of the eae gene encoding intimin β from CR strain ICC169 was amplified by PCR using a forward primer (CReaefor2 5′-CCGTTCTGTCGAATGGTCAAGTAG-3′) and a CR eae β -derived reverse primer overlapping the end of the gene and including an Eag I restriction site (CReaerev1EagI 5′- CGGCCG TACACAGAATTATGGACAGTCCCG-3′).…”

Section: Methodsmentioning

confidence: 99%

“…The DNA fragment between the Sal I site and the 3′ end of the eae gene encoding intimin β from CR strain ICC169 was amplified by PCR using a forward primer (CReaefor2 5′-CCGTTCTGTCGAATGGTCAAGTAG-3′) and a CR eae β -derived reverse primer overlapping the end of the gene and including an Eag I restriction site (CReaerev1EagI 5′- CGGCCG TACACAGAATTATGGACAGTCCCG-3′). The amplified eae fragment, flanked by Sal I and Eag I restriction sites, was used to replace the corresponding fragments of pCVD438 as previously described (Frankel et al , 1998) (Fig. 1).…”

Section: Methodsmentioning

confidence: 99%

“…A third domain (D3) located at the C-terminal tip of the molecule is formed by the 76 amino acid disulfide loop that shows some structural similarity to C-type lectin. The Cys residues (C860 and C937) forming the disulfide loop are totally conserved among the different intimin types and are required for cell-binding activity (Frankel et al , 1995) and A/E lesion formation (Frankel et al , 1998). …”

Section: Introductionmentioning

confidence: 99%

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Functional studies of intimin in vivo and ex vivo: implications for host specificity and tissue tropism

et al. 2007

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Intimin is an outer-membrane adhesin that is essential for colonization of the host gastrointestinal tract by attaching and effacing pathogens including enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR). The N-terminus of intimin from the different strains is highly conserved while the C-terminus, which harnesses the active receptor-binding site, shows sequence and antigenic polymorphism. This diversity was used to define a number of distinct intimin types, the most common of which are α, β and γ. Intimin binds the type III secretion system effector protein Tir. However, a large body of evidence suggests that intimin also binds a host-cell-encoded receptor(s) (Hir), and interaction of different intimin types with Hir contributes to tissue and host specificity. The aims of this study were to compare the activity of the major intimin types (α, β and γ) in vivo and ex vivo, using the CR mouse model and in vitro organ culture (IVOC), and to determine their exchangeability. The results confirm that intimin γ is not functional in the CR mouse model. In the pig, intimin β can substitute for EPEC intimin α but when placed in an EHEC O157 : H7 background it does not produce an intimin α-like tropism, although some adhesion to the small and large intestine was observed. In contrast, in human IVOC, intimin β in an EHEC background produces small intestinal colonization in a similar manner to intimin α.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Functional studies of intimin in vivo and ex vivo: implications for host specificity and tissue tropism

et al. 2007

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“…In addition, the variability in sample properties between donors and results is far greater than in cell line models. The IVOC system has been used to study EPEC-host specificity and tissue tropism (Girard et al 2005;Mundy et al 2007), Tir-intimin-dependent colonization and A/E lesion formation (Frankel et al 1998a;Schüller et al 2007;Frankel and Phillips 2008). The polarized IVOC method has shown that apical EPEC infection of duodenal mucosa results in a flagellin-dependent increase in IL-8 levels (Schüller et al 2009).…”

Section: In Vitro Infection Modelsmentioning

confidence: 99%

In Vitro and In Vivo Model Systems for Studying Enteropathogenic Escherichia coli Infections

Law

Gur-Arie

Rosenshine

et al. 2013

Cold Spring Harbor Perspectives in Medicine

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Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) belong to a group of bacteria known as attaching and effacing (A/E) pathogens that cause disease by adhering to the lumenal surfaces of their host's intestinal epithelium. EPEC and EHEC are major causes of infectious diarrhea that result in significant childhood morbidity and mortality worldwide. Recent advances in in vitro and in vivo modeling of these pathogens have contributed to our knowledge of how EPEC and EHEC attach to host cells and subvert hostcell signaling pathways to promote infection and cause disease. A more detailed understanding of how these pathogenic microbes infect their hosts and how the host responds to infection could ultimately lead to new therapeutic strategies to help control these significant enteric pathogens.

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“…A final line of evidence that suggests that intimin may have a host cell surface receptor other than Tir comes from mutational analysis of the host cell binding domain of intimin-␣. Mutations in this region of the adhesin disrupt the capacity of the bacteria to adhere to the host cell but do not interfere with the in vitro interaction between intimin and Tir (32,33). If Tir functions as the sole intimin receptor, such mutations should not affect bacterial adherence.…”

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confidence: 99%

Cell Surface-localized Nucleolin Is a Eukaryotic Receptor for the Adhesin Intimin-γ of Enterohemorrhagic Escherichia coliO157:H7

Sinclair

O'Brien

2002

Journal of Biological Chemistry

180

145

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Intimin-␥ is an outer membrane protein of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that is required for the organism to adhere tightly to HEp-2 cells and to colonize experimental animals. Another EHEC O157:H7 protein, the Transferred intimin receptor (Tir), is considered the primary receptor for intimin-␥. Nevertheless, Tir-independent binding of intimin-␥ to HEp-2 cells has been reported. This observation suggests the existence of a eukaryotic receptor(s) for intimin-␥. In this study, we sought to identify that receptor(s). First, we determined by equilibrium binding titration that the association of purified intimin-␥ with HEp-2 cells was specific and consistent with a single host cell receptor. Second, we isolated a protein from lysates of HEp-2 cells that bound intimin-␥ and subsequently identified this molecule as nucleolin, a protein involved in cell growth regulation that can be cell surface-expressed. Third, we established that purified intimin-␥ and nucleolin were co-localized on the surface of HEp-2 cells and that the site of EHEC O157:H7 attachment was associated with regions of nucleolin expression. Finally, we demonstrated that mouse anti-nucleolin sera significantly decreased the adherence of EHEC O157:H7 to HEp-2 cells. From this, we conclude that nucleolin is the HEp-2 cell receptor for intimin-␥ expressed by EHEC O157:H7.

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Generation of Escherichia coli intimin derivatives with differing biological activities using site‐directed mutagenesis of the intimin C‐terminus domain

Cited by 57 publications

References 43 publications

Functional studies of intimin in vivo and ex vivo: implications for host specificity and tissue tropism

Functional studies of intimin in vivo and ex vivo: implications for host specificity and tissue tropism

In Vitro and In Vivo Model Systems for Studying Enteropathogenic Escherichia coli Infections

Cell Surface-localized Nucleolin Is a Eukaryotic Receptor for the Adhesin Intimin-γ of Enterohemorrhagic Escherichia coliO157:H7

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