Molecular Microbiology (2002)

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© 2002 Blackwell Science Ltd

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Science, 200245Original Article

H. Liu et al.Point

mutants of EHEC intimin that diminish Tir binding

Accepted 2 July, 2002. *For correspondence. E-mail john.leong@umassmed.edu; Tel. (+1) 508 856 4059; Fax (+1) 508 856 5920.

Point mutants of EHEC intimin that diminish Tir recognition and actin pedestal formation highlight a putative Tir binding pocket

Hui Liu,

1

Padhma Radhakrishnan,

1

Loranne Magoun,

1

Moses Prabu,

2

Kenneth G. Campellone,

1

Pamela Savage,

1

Feng He,

1

Celia A. Schiffer

2

and John M. Leong

1

*

Departments of

1

Molecular Genetics and Microbiology, and

2

Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA.

Summary

Attachment to host cells by enterohaemorrhagic

Escherichia coli

(EHEC) is associated with the forma-tion of a highly organized cytoskeletal structure con-taining filamentous actin, termed an attaching andeffacing (AE) lesion. Intimin, an outer membraneprotein of EHEC, is required for the formation of AElesions, as is Tir, a bacterial protein that is translo-cated into the host cell to function as a receptor forintimin. We established a yeast two-hybrid assay forintimin–Tir interaction and, after random mutagene-sis, isolated 24 point mutants in intimin, whichdisrupted Tir recognition in this system. Analysis of11 point mutants revealed a correlation between rec-ognition of recombinant Tir and the ability to triggerAE lesions. Many of the mutations fell within a 50-residue region near the C-terminus of intimin.Alanine-scanning mutagenesis of this region revealedfour residues (Ser890, Thr909, Asn916 and Asn927)that are critical for Tir recognition. Mapping thesequences of EHEC intimin and Tir onto the crystalstructure of the intimin–Tir complex of enteropatho-genic

E. coli

predicts that each of these four intiminresidues lies at the intimin–Tir interface and contrib-utes to a pocket that interacts with Ile298 of EHEC Tir.Thus, this genetic approach to intimin function bothidentified residues critical for Tir binding and demon-strated a correlation between the ability to bind Tirand the ability to trigger actin focusing.

Introduction

Enterohaemorrhagic

Escherichia coli

(EHEC) hasemerged as one of the most important food-borne patho-gens in North America, Europe and Japan (reviewed inNataro and Kaper, 1998). It is responsible for bothsporadic cases and epidemic outbreaks of haemorrhagiccolitis, and is associated with the haemolytic uraemic syn-drome (HUS), the triad of haemolytic anaemia, thromb-ocytopenia and renal failure. This life-threateningcomplication is due to the high level expression of shiga-like toxins (Stx’s; O’Brien

et al

., 1992) by bacteria thatremain bound to the intestinal mucosa.

Colonization of the intestinal mucosa by EHEC, as wellas by a subset of other intestinal pathogens such asenteropathogenic

E. coli

(EPEC), involves the formationof ‘attaching and effacing (AE) lesions’. Epithelial cellmicrovilli are effaced and bacteria are closely opposed tothe host cell membrane. Most strikingly, filamentous actinis generated beneath the attached bacteria, resulting inthe formation of ‘pedestals’ that lift the bacteria above theplane of the host cell membrane (Staley

et al

., 1969;Ulshen and Rollo, 1980; Moon

et al

., 1983). A contiguous35 kb pathogenicity island, termed the locus of enterocyteeffacement (LEE) is associated with the ability to form AElesions (McDaniel and Kaper, 1997; Perna

et al

., 1998).Included in this locus are genes encoding a type III secre-tion apparatus (for a review, see Hueck, 1998), as well asthe bacterial effector molecules that are translocated bythis apparatus to the host cell (Jarvis

et al

., 1995; Kennyand Finlay, 1995).

A multistep model has been proposed for AE lesionformation (Donnenberg and Kaper, 1992; Donnenberg

et al

., 1997). After initial or ‘non-intimate’ attachment,effector molecules are translocated to the host cell, trig-gering a variety of signalling pathways that result ineffacement of microvilli (Donnenberg and Kaper, 1991;Foubister

et al

., 1994; Ismaili

et al

., 1995; Kenny andFinlay, 1995; Jarvis and Kaper, 1996; Wolff

et al

., 1998).Among the injected proteins is Tir (Kenny

et al

., 1997)(also known as EspE; Deibel

et al

., 1998), which localizesin the host cell membrane. Remarkably, Tir acts as areceptor for the bacterial outer membrane protein intimin,encoded by the

eae

gene (Jerse, 1990; Kenny

et al

.,

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1997). The interaction of Tir and intimin triggers the finalstep of AE lesion formation, leading to actin condensation,pedestal formation and intimate bacterial attachment.Consistent with the hypothesis that this process can occurin discrete steps, laboratory strains of

E. coli

expressingintimin, or even intimin-coated latex beads, form AElesions on mammalian cells that have been preinfectedwith

E. coli

competent for LEE-mediated type III secretion(Rosenshine

et al

., 1996; Liu

et al

., 1999). The observa-tion that disruption of

eae

or

tir

diminishes intestinalcolonization in experimental animals (Donnenberg

et al

.,1993; Marches

et al

., 2000) as well as AE lesion formationindicates an important role for intimate attachment duringinfection.

Several recent studies have shed light on the structureand function of intimin. The crystal structure of the C-terminal extracellular domain of the intimin-related

Yers-inia pseudotuberculosis

invasin protein revealed anelongated 180 Å rod consisting of four domains resem-bling immunoglobulin superfamily (IgSF) domains and afifth domain related to C-type lectin-like domains(Hamburger

et al

., 1999). The last IgSF and the C-typelectin-like domains form a superdomain that is sufficientto bind host cell invasin receptors (a subset of

b

1

-chainintegrins) (Leong

et al

., 1990; Rankin

et al

., 1992).Despite limited sequence identity between invasin andintimin, the global fold of the last 280 amino acids (i.e. lastthree domains) of EPEC intimin, determined by multidi-mensional magnetic resonance (Kelly

et al

., 1999), alsoconsists of two IgSF domains and a C-type lectin-likedomain, and the last two domains were necessary andsufficient for receptor (i.e. Tir) binding (Liu

et al

., 1999).This minimal Tir-binding region of intimin, when purifiedand immobilized on latex beads, was also sufficient totrigger AE lesions on mammalian cells that had beenpreinfected with

E. coli

competent for type III secretion(Liu

et al

., 1999). Nuclear magnetic resonance (NMR)titration analysis of EPEC intimin and Tir implicated a50 aa region at the C-terminus of intimin in Tir recognition(Batchelor

et al

., 2000). Finally, the recently publishedEPEC intimin crystal structure closely matches the struc-ture of invasin, and residues of EPEC intimin that specif-ically contact Tir were identified in intimin–Tir complex(Luo

et al

., 2000).In the current study, we randomly mutagenized the Tir-

binding domain of intimin and isolated point mutants thatdisrupted Tir recognition. The ability of intimin mutants tobind to recombinant Tir correlated with their ability to trig-ger AE lesions on preinfected mammalian cells. Half ofthe mutations fell within the previously identified 50 aa C-terminal region of intimin, and alanine-scanning mutagen-esis of this region identified four residues of EHEC intiminthat are critical for Tir recognition. In a model of the EHECintimin–Tir complex that is based on EPEC intimin and

Tir (Luo

et al

., 2000), these four amino acids are predictedto be located at the intimin–Tir interface, indicating thatthese residues play a functional role in intimin recognitionby Tir.

Results

Optimization of two-hybrid assay to detect Tir–intimin interactions

To identify residues of intimin that are critical for Tir bind-ing, and to begin to examine potential affinity require-ments of intimin–Tir interactions in the generation of AElesions, we sought to isolate mutations in intimin thatdiminished Tir recognition. The yeast two-hybrid systemcan provide a simple screen for mutations that diminishprotein–protein interactions, so we first optimized a two-hybrid assay for intimin-Tir binding. Full-length Tir wasfused to the GAL4 activation domain in the yeast two-hybrid vector pGAD424, generating a fusion protein des-ignated Gal4 AD-Tir. The C-terminal 395 aa of intimin,which probably comprise the entire extracellular domainof intimin (Hamburger

et al

., 1999; Batchelor

et al

., 2000;Luo

et al

., 2000), were fused to the LexA DNA-bindingdomain in a yeast two-hybrid vector to generate fusionprotein LexA–Int395 (Fig. 1A; see

Experimental proce-dures

). Yeast transformants expressing LexA–Int395 andGal4 AD–Tir produced

ª

27 Miller units of

b

-galactosidaseactivity, i.e. approximately 10-fold greater than thebackground levels determined by control transformantsexpressing a LexA fusion to the yeast protein NMD2 (He

et al

., 1996) and Gal 4 AD–Tir (Fig. 1A). The C-terminal181 aa of intimin are sufficient to bind recombinant Tir(Liu

et al

., 1999; Batchelor

et al

., 2000), and a yeast strainexpressing LexA–Int181 and Gal4 AD–Tir producedhigher levels of

b

-galactosidase activity (

ª

400 Millerunits). The C-terminal 100 residues of intimin are devoidof Tir binding activity, and as expected, coexpressionof LexA–Int100 with Gal4AD–Tir did not result in

b

-galactosidase activity above background levels. The twotransmembrane domains of Tir (Kenny

et al

., 1997) dividethis protein into three intervals, an N-terminal (TirN), acentral (TirM) and C-terminal region (TirC) (Fig. 1B). Theintimin-binding region of EPEC Tir has been mapped tothe central domain, TirM (de Grado

et al

., 1999; Hartland

et al

., 1999; Kenny, 1999). To optimize signal strength inthe two-hybrid assay, GAD–Tir fusion proteins carryingone or more of these Tir domains were generated andcoexpressed with LexA–Int181 (Fig. 1B). As expected,expression of fusions carrying only the N-terminal(TirN) or C-terminal (TirC) regions gave background

b

-galactosidase activity (determined by coexpression ofLexA–Int181 and the Gal4 AD fused to the yeast proteinUPF3 (He

et al

., 1997; Fig. 1B). Yeast transformants coex-

Point mutants of EHEC intimin that diminish Tir binding

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pressing Gal4 AD fusions carrying the central region ofTir (TirN + M + C, TirM + C, or TirM) and LexA–Int181resulted in

b

-galactosidase activity at least 100-fold abovebackground levels. The smallest fusion, Gal4 AD–TirM,gave the highest

b

-galactosidase activity, giving rise todark blue colonies on Xgal plates and almost 2000 Miller

units, some 600-fold higher than background levels. Theintimin mutant V906A diminishes Tir binding (Liu

et al

.,1999), and when this mutation was generated inLexA–Int181, the two-hybrid signal was diminishedapproximately fourfold (Fig. 1C). Yeast transformantsexpressing LexA–Int100, which is lacking a complete

Fig. 1.

Establishment of a yeast two-hybrid assay for enterohaemorrhagic

Escherichia coli

(EHEC) intimin–Tir interaction.A. The minimal Tir-binding region of intimin interacts with Tir in the yeast two-hybrid assay. The regions of intimin responsible for outer membrane localization (‘OM Localization’) or Tir-binding are indicated on protein map. Depicted below map are the LexA–intimin fusions carrying the indicated regions of intimin (shaded bar) that were coexpressed with GAL4 activation domain fused to full-length Tir (‘Gal4 AD–Tir’) in the yeast reporter strain L40. (Number in intimin fusion name indicates the number of residues of C-terminus included in each fusion.) The LexA fusion to the yeast protein NMD2 (He

et al

., 1997) served as a negative control. For each LexA fusion, the mean (

±

SD)

b

-galactosidase activity of three transformants is represented.B. Gal4 AD fused to the central domain of Tir (TirM) gives a strong two-hybrid signal with LexA–Int181. Depicted on map are the N-terminal (‘N’), middle (‘M’), and C-terminal (‘C’) segments of Tir, separated by transmembrane domains (‘TM’). Depicted below map are the Gal4 AD–Tir fusions carrying the indicated regions of Tir that were coexpressed with LexA–Int181 (see ‘A’). A Gal4 fusion to the yeast protein UPF3 (He

et al

., 1997) served as a negative control.C. An intimin mutation known to diminish Tir interaction results in a diminished two-hybrid signal. Wild-type LexA–Int181, or a derivative carrying the V906A mutation (‘Int181–V906A’) that was previously shown to disrupt intimin–Tir interaction (Liu

et al

., 1999), was coexpressed with Gal4 AD-TirM. LexA fused to the C-terminal 100 amino acids of intimin, or to the yeast protein NMD2 (He

et al

., 1997) served as negative controls.D. The two-hybrid signal generated by coexpression of LexA–intimin and GAD–Tir reflects a direct interaction between intimin and Tir. Various MBP–intimin fusion proteins were purified and separated by 10% SDS-PAGE. Top panel shows Coomassie blue-stained gel, and bottom panel shows binding of filter replica by GST–TirM (see

Experimental procedures

). ‘

b

-gal’, MBP-

b

-galactosidase; ‘Int395’, MBP-Int395; ‘Int-395C-S’, MBP-Int395 C932S mutant; ‘Int277’, MBP-Int277; ‘Int181 V-A’, MBP-Int181 V906A; ‘Int181’, MBP-Int181; and ‘Int100’, MBP-Int100.

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Tir binding domain, produced only background levels of

b

-galactosidase activity when coexpressed with Gal4AD–TirM (Fig. 1C).

To confirm that the transcriptional activation detected inthe yeast two-hybrid system reflected a direct interactionbetween EHEC intimin and Tir, various MBP–intiminfusion proteins were tested for binding to purified GST–TirN, GST–TirM or GST–TirC in gel overlay assays. OnlyGST–TirM demonstrated intimin-binding activity (Fig. 1Dand data not shown), and this binding, as predicted,required the C-terminal 181 aa of intimin, because fusionprotein MBP-Int100 carrying only the last 100 aa of intiminshowed no binding activity (Fig. 1D, ‘Int100’). Disruptionof a 76-amino-acid disulphide loop in intimin (Kelly

et al

.,1999; Luo

et al

., 2000) resulted in loss of GST–TirM rec-ognition (Fig. 1D, ‘Int395C-S’), as did the V906A mutationpreviously shown to disrupt Tir binding (Liu

et al

., 1999)(Fig. 1D, ‘Int181V-A’). These data indicate that the centralregion of EHEC Tir directly interacts with intimin, in parallelwith Tir–intimin interactions of EPEC (de Grado

et al

.,1999; Hartland

et al

., 1999; Kenny, 1999), and suggestthat the yeast two-hybrid assay reflects this interaction.

Intimin mutants that disrupt Tir-binding in a yeast two-hybrid assay

We next used the yeast two-hybrid assay described aboveto identify intimin mutants that were diminished in Tir-binding. Although coexpression of LexA–Int181 (frompLexA–Int181) and Gal4 AD–TirM resulted in dark bluecolonies on Xgal plates, about 500 white or light bluecolonies were identified upon screening approximately25 000 yeast colonies after random mutagenesis of theintimin coding region of pLexA–Int181 (see

Experimentalprocedures

). Whole-cell extracts from these selectedyeast colonies were immunoblotted using an anti-LexAmAb to identify strains that express full-length LexA–Int181 fusion protein. A total of 24 plasmids that expressfull-length fusion protein were characterized by sequenc-ing their respective intimin coding regions, revealing 16single-site mutants, six double-site mutants and two triple-site mutants (Fig. 2). Plasmids encoding most of theseLexA–Int181 point mutants were retransformed into ayeast tester strain expressing Gal4 AD-TirM, and

b

-galactosidase activity was measured (see

Experimental

Fig. 2.

Point mutants within Int181 that disrupt Tir binding in a yeast two-hybrid assay. The intimin-encoding region of a plasmid expressing LexA–Int181 was randomly mutagenized. Point mutants that still produce full-length fusion protein but give diminished

b

-galactosidase activity when coexpressed in a yeast reporter strain with Gal4 AD–TirM were identified (see text). The

b

-galactosidase activity of each strain was quantitated, and the percentage of wild-type activity is given. The region of intimin that was subsequently subjected to alanine scanning mutagenesis is indicated. ‘ND’, not determined.

Point mutants of EHEC intimin that diminish Tir binding

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procedures

) (Fig. 2). The mutants gave rise to

b

-galactosi-dase activity 0.5–7% of the levels expressed by transfor-mants harbouring pLexA–Int181. 70% (17/24) of themutations altered residues that were completely con-served among the intimin sequences from EHECO157:H7, EPEC E2348/69, and

Citrobacter rodentium

.Given that these sequences share only 36% identity overthe entire C-terminal 181 amino acids, these results sug-gest that the mutations generated using this yeast two-hybrid system identify functionally critical residues.

A subset of intimin mutations that diminish Tir binding in the yeast two-hybrid assay also diminish Tir binding by full-length intimin expressed in

E. coli

To investigate the effect of these intimin mutations whenthe protein was expressed at relatively high levels on thesurface of

E. coli

, we generated 14 of the single-sitemutations in full-length

eae

on a high copy number plas-mid (see

Experimental procedures

). MBP-Int395 carryingthe mutation C932S lacks the disulphide loop at intiminC-terminus (Kelly

et al

., 1999; Luo

et al

., 2000) and isdefective at binding GST–TirM (Fig. 1D). Full-lengthintimin with the identical mutation was generated andincluded in this analysis as a binding-incompetent control.Pilot experiments revealed that expression of intimin fromhigh-copy number plasmid in the EHEC strain EDL933 didnot result in intimin easily detectable on the bacterialsurface (data not shown). Therefore, the intimin mutantswere expressed in the

E. coli K-12 strain MC1061.For each intimin mutant, we first determined the level

of intimin expression on the bacterial surface level, byprobing intact, immobilized bacteria with anti-intimin anti-serum in a modified enzyme-linked immunoabsorbanceassay (ELISA) (Liu et al., 1999; see Experimental proce-dures). Four of the mutants gave ELISA signals at levelsat least fivefold lower than the wild type (Table 1).Although we cannot rule out the possibility that thediminished ELISA signal of these mutants is due to dimin-ished antibody affinity, the simplest interpretation, giventhe polyclonal nature of the antiserum, is that these muta-tions disrupt the folding and/or secretion of full-lengthintimin. One of the residues altered in these mutants,W894, is equivalent to a residue in EPEC intimin (W899)that is located in the interior of the protein and is requiredfor intimin function (Batchelor et al., 2000). The other 11intimin mutants gave ELISA signals at a level at least 44%of the wild type (Table 1).

To determine if the Tir-binding defects of intimin muta-tions apparent in the yeast two-hybrid assay were alsoobserved when full-length intimin was expressed on thebacterial surface, the ability of bacteria expressing thesemutants to bind to GST-Tir was determined (see Experi-mental procedures). As expected, intimin mutants that

were poorly expressed on the bacterial surface did notbind to GST–Tir significantly above background levels(Table 1). Mutants that were expressed on the bacterialsurface demonstrated a wide range of GST-Tir bindingactivity. Three mutations, including the C932S mutationthat disrupts the C-terminal disulphide loop, abolishedGST–Tir binding in this assay. One of the residues altered,W796, is equivalent to a residue of EPEC intimin (W795)that was previously shown to be required for full EPECintimin function (Reece et al., 2001). Three mutants,Q902L, V845A, and V931D, bound GST-Tir at near(≥88%) wild-type levels. The remaining five mutantsbound GST–Tir between 45% and 83% of wild-type levels.Thus, although all of the intimin mutations appeared todisrupt Tir recognition in the yeast two-hybrid assay, mostof the mutations did not severely diminish binding ofGST–Tir by bacteria expressing relatively high levels offull-length intimin.

The ability of intimin-expressing bacteria to bind to GST–Tir correlates with the ability to trigger AE lesions on mammalian cells preinfected with EPEC

To test the hypothesis that Tir-binding is the essentialactivity of intimin in triggering the final step of AE lesionformation, we tested whether these intimin mutants couldtrigger the last step of AE lesion formation. E. coli K12expressing EHEC intimin from a high-copy number plas-mid is able to generate robust AE lesions on mammaliancells preinfected with JPN15.96, an EPEC eae mutant thatretains the ability to secrete effector molecules (includingTir) via the type III machinery (Jerse, 1990). This obser-vation suggested that the Tir-binding domain of EHECintimin, which is 47% identical to that of EPEC intimin(Perna et al., 1998), is able to recognize EPEC Tir effi-ciently enough to trigger AE lesions, a hypothesis that issupported by direct binding studies (DeVinney et al.,1999). (EHEC form AE lesions in vitro much less effi-ciently than EPEC (Cantey and Moseley, 1991), andpreinfection of monolayers with EHEC did not result inAE lesions upon subsequent challenge with E. coli K12expressing intimin; data not shown).

We assessed both the quantity of AE lesions generatedby the mutants (by counting the number of AE lesionsformed per 100 mammalian cells) and the quality of thoselesions (by visually scoring the strength of phalloidin stain-ing of F-actin) (Table 1; see Experimental procedures).This analysis revealed a general correlation between Tirbinding activity and AE lesion formation. As expected,mutants with virtually no GST–Tir binding activity did nottrigger AE lesions (Table 1) and, in fact, were not able tobind efficiently to the preinfected monolayers (Fig. 3).Mutants that retained the ability to bind GST–Tir at nearwild-type levels (Q902L, V845A and V931D) triggered AE

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lesions indistinguishable in quantity or quality from wildtype. Four out of the five remaining mutants, which wereonly moderately defective for binding GST-Tir (45% to83% wild-type levels) triggered AE lesions less robust inphalloidin staining than wild type (Table 1, Fig. 4). Threeof these mutants also triggered significantly fewer AElesions, and their efficiency of AE lesion formation corre-lated with their respective GST–Tir binding activities. Oneout of the five mutants, Y882C, demonstrated a partialdefect in GST-Tir binding (63% of wild-type levels), but hadno apparent defect in AE lesion formation. One potentialexplanation for this result is that the mutation may alterintimin binding to a soluble, recombinant fragment ofEHEC Tir (GST–TirEHEC) more significantly than it doesbinding to native EPEC Tir localized in the eukaryotic

membrane (see also Discussion). We conclude that rela-tively mild defects in GST–Tir binding when intimin isexpressed at high levels on the bacterial surface canresult in significant defects in AE lesion formation. In addi-tion, the general correlation between GST–Tir binding andactin pedestal formation on preinfected cells suggeststhat Tir binding activity is the critical property of intiminrequired to trigger the final step of AE lesion formation.

Alanine scanning mutagenesis reveals that intimin residues S890, T909, N916 and N927 are critical for Tir binding

Eight out of the 16 single mutations of intimin that dis-rupted Tir binding in the yeast two-hybrid assay altered

Table 1. Tir-binding by intimin generally correlates with the ability to trigger AE lesions.

Intimin expresseda Surface expressionb GST-TirEHEC bindingc

AE lesions on Hep-2 cells preinfected with EPECd

Lesions/100 cellse Actin condensationf

Wild-type 1.00 1.00 32.6 ± 1.6 ++None 0.00 0.00 0 NAg

Intimin expressed poorly on the bacterial surfaceb

N911I 0.20 ± 0.06 0.05 ± 0.02 0 NAP839L 0.00 ± 0.01 0.00 ± 0.01 0 NAW894R 0.06 ± 0.02 0.00 ± 0.01 0 NAS826P 0.14 ± 0.07 -0.03 ± 0.10 0 NA

Intimin expressed well on the bacterial surfaceb

Q902L 0.78 ± 0.09 1.23 ± 0.09 27.7 ± 2.2 ++V845A 0.67 ± 0.06 0.94 ± 0.15 30.5 ± 2.8 ++V931D 0.89 ± 0.07 0.88 ± 0.05 27.6 ± 2.2 ++V906E 0.53 ± 0.06 0.69 ± 0.11 23.9 ± 3.0* +Y882C 0.85 ± 0.03 0.63 ± 0.02 29.7 ± 2.7 ++V928I 0.44 ± 0.04 0.58 ± 0.04 29.2 ± 3.5 +P863L 0.98 ± 0.02 0.56 ± 0.01 13.8 ± 1.1* ±N927D 0.78 ± 0.01 0.45 ± 0.03 6.4 ± 0.5* ±W796R 0.68 ± 0.05 0.05 ± 0.01 0 NAG781D 0.78 ± 0.07 0.03 ± 0.01 0 NAC932S 0.85 ± 0.03 -0.02 ± 0.01 0 NA

a. Full-length intimin derivatives expressed in E. coli K12 strain MC1061 from a high copy number (pUC19-derived) plasmid.b. The expression of intimin on the bacterial surface was determined by enzyme-linked immunoabsorbance assay (ELISA) using anti-intiminantiserum, and was normalized to the level of expression of wild-type intimin (see Experimental procedures). Mutants that expressed intimin atlevels 20% or less than that of wild type are grouped separately from those that expressed intimin at levels at least 50% of wild type. Shown arethe mean and standard deviation of four determinations.c. Bacteria were tested for the ability to bind to GST–TirEHEC, as determined by a modified ELISA assay using anti-GST antiserum. Values werenormalized to the level of Tir-binding by wild-type intimin (see Experimental procedures). Shown are the mean and standard deviation of fourdeterminations.d. HEp-2 cells were preinfected with an enteropathogenic Escherichia coli (EPEC) eae mutant, and the indicated bacterial strain was added tothese preinfected monolayers at a multiplicity of infection (moi) of approximately 5. Each sample was coded and then scored blindly for attachingand effacing (AE) lesions after staining infected monolayers with TRITC-phalloidin (see Experimental procedures).e. Attaching and effacing lesions were counted on at least 100 HEp-2 cells, and the number of AE lesions per 100 cells calculated. During eachexperiment, mutants were tested in triplicate. Each mutant was analysed in six independent experiments, and shown are the mean and standarderror of the resulting 18 determinations. Asterisks indicate p < 0.05 for the difference between E. coli expressing mutant or wild-type intimin inthe number of AE lesions/100 cells, as determined by the Mann–Whitney U-test.f. Each strain was visually scored (in a blinded fashion) for the degree of phalloidin staining of AE lesions. ‘++’, indistinguishable from wild type,with nearly all lesions showing strong phalloidin staining; ‘+’, most AE lesions strongly stained, some AE lesions weakly stained; ‘±’, all AE lesionsweakly stained. Each mutant was scored three times per experiment, and each experiment was performed six independent times. The consensusscore shown was obtained in at least 12 out of the 18 determinations.g. NA, not applicable.

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residues in a 50-amino-acid interval, residues 882–931(Fig. 2). These included the two most conservative substi-tutions, N927D and V928I, and a mutation of V906, aresidue that was implicated previously as a residue critical

for Tir binding (Liu et al., 1999). In contrast, all of the eightmutations that fell outside of this 50-residue region werenon-conservative substitutions. The 50-amino-acid C-terminal region also contains residues with amide reso-nances that move or broaden in the presence of Tir, impli-cating them in Tir recognition (Batchelor et al., 2000). Wetherefore subjected residues in this region to alaninescanning mutagenesis. At the time that these mutantswere generated, 33 intimin sequences had been identifiedand the crystal structure of the Y. pseudotuberculosisinvasin had been published (Hamburger et al., 1999). Wesubstituted alanine for 10 amino acids in this interval thatfulfilled the following criteria. First, these residues wereconserved among at least 27 out of the 33 sequencedmembers of the intimin family (Yu and Kaper, 1992;Beebakhee et al., 1992; An et al., 1997). Second, thesewere residues for which alanine was not found in any ofthe sequenced intimin proteins. Third, to target residuesmore likely to be on the surface of intimin, we did notmutate codons specifying aliphatic or aromatic aminoacids, or residues that corresponded to buried residues inY. pseudotuberculosis invasin protein. To facilitate a bio-chemical analysis of Tir binding by the mutants, we con-structed all of the mutations on the plasmid that encodedMBP-Int277, a maltose binding protein fusion that con-tains the C-terminal 277 amino acids of EHEC intimin (Liuet al., 1999). Three out of the 10 mutants (E901A, N911A,and T914A) could not be purified (data not shown), sug-gesting that these mutations cause misfolding of theprotein and subsequent degradation. The other sevenmutants demonstrated a variable degree of degradationproducts after purification (Fig. 5A), but were purified insufficient quantities to permit biochemical analysis.

Fig. 4. Point mutants of intimin that diminish Tir recognition diminish AE lesion formation. HEp-2 cells were preinfected with the EPEC eae mutant JPN15.96 and then incubated with gen-tamicin to kill bacteria (see Experimental pro-cedures). Monolayers were then infected with E. coli K12 strains expressing either the intimin point mutant N927D (A–C) or wild-type intimin (D–F). Cell associated bacteria, which express GFP, were detected under phase contrast (A and D) or fluorescence (B and E) microscopy. Filamentous actin was detected using TRITC-labelled phalloidin (C and F). Arrowheads indi-cate cell-bound bacteria and associated fila-mentous actin.

Fig. 3. Intimin mutants that are defective for Tir binding do not bind to mammalian cells preinfected with EPEC. HEp-2 cells were mock infected or preinfected with the EPEC eae mutant JPN15.96 and then incubated with gentamicin to kill bacteria (see Experimental proce-dures). Monolayers were then infected with E. coli K12 strains expressing wild-type or mutant intimin and, after washing, bound bacteria were quantitated by plating on bacteriological media. (E. coli expressing intiminC932S was tested in a separate experiment than the other two mutants; in that experiment, 9 ± 1.7% of E. coli express-ing wild-type intimin bound to preinfected monolayers.) The mean ± SD of triplicate samples is shown.

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We examined the Tir binding activity of each of thepurified mutants by a gel overlay assay, probing filters withrecombinant GST-TirM and revealing bound GST-Tir withanti-GST antibody. At least three mutants, S890A, T909A,and N927A, were diminished in Tir binding (Fig. 5A). Toobtain a more quantitative assessment of defects in Tir-binding by the intimin mutants, microtitre wells coated withwild-type or mutant MBP-intimin fusion proteins wereprobed with recombinant TirM. The same three mutantsdemonstrated severe (p < 0.0001) defects in Tir recogni-tion in this assay (Fig. 5B). In addition, Tir-binding byN916A was partially but significantly (p = 0.0001) dimin-ished, i.e. demonstrated binding at least 21% comparedwith that of wild-type MBP-intimin in repeated experiments(Fig. 5B and data not shown).

To determine the likely location of the above residueswith respect to the intimin–Tir binding interface, we

mapped the sequences of EHEC intimin and Tir onto theknown crystal structure of the EPEC intimin–Tir complex(Fig. 6A; Luo et al., 2000); see Experimental procedures).The extracellular domains of EPEC and EHEC Tir are72% identical and the Tir binding domains of EPEC andEHEC intimin are 47% identical, and the sequence align-ments can be made without introducing any insertions (Yuand Kaper, 1992; Elliott et al., 1998; Perna et al., 1998).Thus, the resulting model in which aligned residues areswapped should be physically reasonable. Analysis of thealanine scanning data in terms of this model shows thatQ897A and S886A, which have no discernible bindingphenotype, are remote from the presumed binding inter-face. T892A, which also exhibits no binding phenotype, isin the middle of the interface. Although the alanine sub-stitution for the larger residue threonine probably resultsin a small indentation on the intimin surface, sequencemapping analysis suggests that T892 is not in the vicinityof potential hydrogen bonding partners and does notmake van der Waal’s contact with Tir. Thus, it is reason-able that T892A displayed no detectable Tir bindingdefect.

The four residues that were identified as critical for Tirbinding, S890, T909, N916 and N927, are predicted toform a small groove that surrounds the side chain of TirI298 (Fig. 6). Intimin residue T909 (along with residuesT892, V921 and V928) is at the base of the putativebinding groove, whereas S890, N916 and N927 surroundthe edge. N927 is predicted to make the closest contactwith Tir, consistent with the observation that the intiminmutant N927D that was isolated from our initial screenwas highly defective for promoting AE lesion formationwhen expressed on the bacterial surface (Table 1). Thus,the intimin residues critical for Tir binding identified by thisgenetic approach are likely to directly interact with Tir, andthe location of these residues specifically highlight a puta-tive Tir binding groove in which Tir residue I298 fits.

Discussion

One of the goals of this study was to identify residues ofEHEC intimin that are critical for Tir binding. For thispurpose, we isolated mutants that were defective forTir-binding in a yeast two-hybrid assay after randommutagenesis of intimin’s Tir binding domain. Half of themutations, including the most conservative substitutions,fell within a 50-residue C-terminal region of intimin previ-ously implicated in Tir binding by NMR analysis (Batcheloret al., 2000). Within this region, we mutated 10 residues,chosen on the basis of their conservation among mem-bers of the intimin family and their likelihood, by compar-ison to the structure of invasin, to be located on the proteinsurface. Alanine substitution of four of these residuessignificantly diminished Tir binding. By mapping the

Fig. 5. Alanine-scanning mutagenesis reveals that intimin residues S890, T909, N916 and N927 are critical for Tir recognition.A. The indicated MBP-intimin fusion proteins were subjected to SDS-PAGE and Coomassie stained (top panel; ‘Coomassie stained’) or transferred to a filter membrane and probed with GST–TirM (bottom panel; ‘GST-TirM blot’; see Experimental procedures). Bound GST–TirM was revealed using anti-GST antibody. Full-length fusion protein is indicated by arrow. (Note that the weak GST–TirM binding by MBP-IntQ897A corresponds to a lesser amount of protein loaded compared with the other MBP–intimin fusion proteins.)B. Microtitre wells coated with the indicated MBP–intimin fusion pro-tein were probed with GST–TirM, and Tir binding was quantitated by ELISA using anti-GST antibody (see Experimental procedures). (Shown is a composite of two experiments, normalized by the inclu-sion of S886A in both experiments.) Brackets indicate 95% confi-dence values of quadruplicate samples. Asterisks indicate binding is significantly different from wild-type control (two-tailed t-test, p £ 0.0001).

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sequence of EHEC intimin and Tir onto the recently deter-mined EPEC intimin–Tir complex crystal structure (Luoet al., 2000), these four amino acids are likely to be a partof a Tir-binding groove on the surface of intimin. In con-

trast, residues that did not alter Tir binding upon alaninesubstitution are predicted either to not interact with Tir, orto interact only through backbone interactions that maynot be altered upon alanine substitution. These results,

Fig. 6. Intimin residues identified by alanine substitution that disrupt the putative intimin–Tir interface. The sequence of intimin and Tir from EHEC was mapped onto the crystal structure of the intimin–Tir complex from EPEC. Intimin is shown in cyan with essential interface residues Ser 890, Thr 909 and Asn 927 in magenta, and slightly less important Asn916 shown in orange. Tir is shown in white.A. Space-filling CPK model of the intimin–Tir complex.B. Space-filling CPK model of intimin showing the binding interface.C. A close up backbone view of the intimin–Tir binding interface. The residues Ser890, Thr909, Asn916 and Asn927 from intimin, and Asn297, Ile298 and Asn303 from Tir are explicitly shown. Oxygen and nitrogen atoms are highlighted in red and blue respectively. The central Ile298 of Tir is highlighted with a van der Waal’s surface.

A B

C

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plus the fact that the Tir-binding region of intimin and theintimin-binding region of Tir are both highly conservedbetween EPEC and EHEC, provide support for the accu-racy of the mapping of the EHEC sequence onto theEPEC intimin–Tir complex crystal structure. Consistentwith this assertion, an alanine substitution mutant ofEPEC intimin (I897A) that is equivalent to the EHECintimin T892A shown here to retain Tir binding activity,also bound Tir indistinguishably from wild-type (Reeceet al., 2001).

The ultimate identification of critical intimin residuesvalidates the use of the yeast two-hybrid screen for thispurpose, but this assay system did not uniformly reflectTir-intimin binding by other assays. For example, most ofthe residues that were identified by the two-hybrid screen(i.e. all except V906, N911 and N927) are predicted tobe far from the binding interface (Luo et al., 2000); P.Radhakrishnan, M. Prabu and C. Schiffer, unpublishedobservations), and mutation of some of these had littleeffect on binding of intimin to recombinant Tir in vitro oron triggering actin pedestals on cells preinfected withEPEC. Furthermore, inclusion of regions of Tir or intiminoutside of their interacting domains reduced the signal inthe two-hybrid assay, suggesting that the size of the fusionproteins and/or their ability to localize to and fold properlyin the yeast nucleus are likely to have large effects in thisassay system. Consistent with the idiosyncratic nature ofthe two-hybrid assay system for Tir–intimin interactions,two-hybrid signals for EPEC intimin and Tir are relativelyweak (de Grado et al., 1999; Hartland et al., 1999).

Although Tir recognition by EPEC and EHEC intimin isclearly similar, some equivalent mutants in these twoproteins display apparently discrepant phenotypes.Whereas no Tir-binding defects in EPEC intimin mutantsT914A and C937S were detected in two-hybrid or geloverlay assays (Hartland et al., 1999; Reece et al., 2001),the corresponding EHEC mutants T909A and C932Sdescribed here were defective for Tir binding in gel over-lay or modified ELISA assays, or both (Figs 1D and 5). Inaddition, bacteria expressing EHEC intimin C932S boundneither to recombinant EHEC Tir (Table 1), nor to mono-layers expressing EPEC Tir in the eukaryotic membraneby virtue of preinfection with EPEC (Fig. 3). Interestingly,the C932S mutation showed virtually no defect in Tir-binding in the yeast two-hybrid assay (H. Lui, unpublishedobservations), indicating that the apparent degree of Tirbinding activity retained by an intimin mutant can varywith the assay type and with the particular form of intiminderivative. Supporting this notion, recombinant proteinscarrying the extracellular domain of intimin display bind-ing activities that are not detectable for full length intiminexpressed on the bacterial surface (Deibel et al., 2001).

Although we cannot rule out the possibility that the appar-ently non-identical Tir binding phenotypes of correspond-ing EPEC and EHEC intimin mutants are solely due todifferences in the intimin and Tir sequences of the twopathogens, it is possible that EPEC Int T914A and C937Shave Tir binding defects that are not detectable in someassays, for example, two-hybrid or gel overlay assays(Hartland et al., 1999; Reece et al., 2001). EPEC IntC937S or IntT914A do not show the full range of biologi-cal activity of intimin, and it has been postulated that thisfailure is due to their inability to bind an unidentifiedendogenous host cell receptor (Hartland et al., 1999;Reece et al., 2001). An alternative interpretation is thattheir phenotypes may be due to (relatively subtle) Tirbinding defects of IntT914A and C937S that may beapparent only when these mutant proteins are expressedat natural levels on the bacterial surface. The recent iden-tification of nucleolin as a putative endogenous host cellreceptor for EHEC intimin may provide an opportunityto experimentally test these possibilities (Sinclair andO’Brien, 2002).

A second goal of this study was to assess the relation-ship between the Tir binding activity of intimin and itsability to trigger the final step of AE lesion formation. Theeae mutations derived from the two-hybrid screen, whenexpressed as full-length intimin protein on the surface ofE. coli K12, varied widely in their ability to bind to recom-binant Tir. We found that relatively subtle defects in theability to bind recombinant Tir (e.g. ªtwo-thirds of wild-typelevels) were associated with significant defects in AElesion formation, as measured by the number and inten-sity of actin pedestals visualized by phalloidin staining ofF-actin. Additionally, binding of intimin to recombinant Tircorrelated with the ability to trigger actin condensation.Mutant Y882C was the sole exception, being moderatelydefective for binding recombinant Tir but showing nodefect in actin signalling. There are several possible expla-nations for this last result, including the fact that we usedrecombinant EHEC Tir to assay binding but, by necessity,used EPEC (not EHEC) for preinfection of mammaliancells to assay actin pedestal formation. Although theintimin binding domains of EHEC and EPEC Tir are 72%identical and each has the capacity to recognize the het-erologous intimin (DeVinney et al., 1999), it is possiblethat they differ significantly in their ability to recognize theintimin Y882C mutant. This last exception notwithstand-ing, we believe these results support the notion that Tirbinding is the primary activity necessary to trigger AElesions after E. coli type III secretion. It is possible thatintimin’s sole function in actin signalling is to cluster Tir inthe plasma membrane of the eukaryotic cell. Furtherinvestigation is required to clearly define the activities of

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intimin that are critical for colonization by attaching andeffacing pathogens.

Experimental procedures

Bacterial and tissue culture

All E. coli strains were grown in L broth at 37∞C, supplementedwith 100 mg ml-1 of ampicillin when appropriate. HEp-2 cellswere cultured in RPMI 1640 (Gibco-BRL) supplementedwith 7% fetal bovine serum and 100 U ml-1 of penicillin,100 mg ml-1 of streptomycin, and 2 mM L-glutamine.

Construction of GAL4(AD)–Tir fusions and LexA(DB)–intimin fusions

Genomic DNA from EHEC O157:H7 strain EDL933 was puri-fied as described previously (Pooyan et al., 1994). Oligonu-cleotides used for amplification of tir and eae were generatedby Operon Technologies, or Life Technologies (see list inTables 3 and 4). DNA segments encoding Tir derivativeswere amplified by Pfu Turbo PCR system (Stratagene) andinserted into pGAD424 to generate GAL4(AD)–Tir fusions(Table 1). DNA fragments encoding intimin C-terminal deriv-atives were amplified as above and inserted into pBTM116to generate LexA(DB)–Tir fusions (Table 1).

Yeast transformation

Yeast strain L40 (MATa his3D200 trpI-901 leu2–3112 ade2LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lacZ GAL4 gal80), inwhich the lacZ and HIS3 promoters are fused to multimer-ized LexA binding sites (Hollenberg et al., 1995), was usedas the reporter strain in the yeast two-hybrid system. Thisstrain can detect weak LexA activators as histidine prototro-phs without the use of 3-aminotriazole. L40 was co-transformed with pGAD424- and pBTM116-derived plasmidsby using the standard LiAc transformation procedure. Briefly,a single colony of L40 was inoculated into 3 ml of yeastextract peptone dextrose (YEPD) medium and grown at 30∞Covernight with shaking. The overnight culture was thendiluted 1:50 into 50 ml of YEPD medium, bringing the OD600

to approximately 0.2, then grown at 30∞C with shaking untilOD600 reached 0.4–0.6. The cells were harvested by centrif-ugation at 1225 g for 5 min. The cells were washed once insterile distilled water and resuspended in 250 ml of sterile 1¥LiAc/TE (0.1 M LiAc, 0.01 M Tris, pH 7.5, 1 mM EDTA). Then,0.3 mg of each plasmid DNA and 100 mg of herring testescarrier DNA (Promega) were added to 100 ml of the aboveyeast competent cells. Next, 600 ml of sterile 40% PEG/0.1 MLiAc/TE (40% PEG4000 in 0.1 M LiAc/TE) was added to themixture and mixed by vortexing. The cells were incubated at30∞C for 30 min with shaking. Then, 70 ml DMSO (SigmaChemical Co.) was added to the cells, and the cells wereheat-shocked at 42∞C for 15 min followed by incubationon ice for 1–2 min. The cells were harvested by centrifuga-tion at 14 000 r.p.m. in a microcentrifuge, resuspended in500 ml of sterile distilled water, and plated onto -Trp -Leu

synthetic dropout (SD) plates to select for the desiredco-transformants.

Quantitation of b-galactosidase activity by ONPG liquid assays

Individual colonies of yeast transformants were inoculatedinto 4 ml of SD-Trp-Leu medium and grown at 30∞C withshaking until mid-log phase. Cells were harvested, washedin sterile distilled water and resuspended in 2 ml of Z-buffer(0.1 M NaPO4, pH 7.00, 10 mM KCl, 1 mM MgSO4) plus0.27% 2-mercaptoethanol. The optical density of the cellsuspension at 600 nm was determined (A600). Then, 200 mlof the cell suspension was added to a microcentrifuge tubecontaining 300 ml of Z-buffer, 50 ml of chloroform and 50 ml of0.05% SDS. The cells were then permeablized by vortexmixing at high speed for 30 s. A 200 ml aliquot of the o-nitrophenyl-b-D-galactoside (ONPG) substrate in H2O(4 mg ml-1) was added and the reaction mixture was incu-bated at 30∞C until the colour yellow was sufficiently devel-oped. The reaction was then stopped by the addition of 0.2 mlof 1 M Na2CO3. The optical density of the o-nitrophenolproduct was measured at 420 nm (A420). Units of the b-galactosidase activity were defined as (A420 ¥ 1000)/[A600 ¥volume (in ml) ¥ time (in min)], as described (Miller, 1972).

Random mutagenesis of the Tir-binding region of eae in a yeast two-hybrid plasmid

To randomly mutagenize the C-terminal 181 codons of eaein the LexA(DB)–intimin fusion plasmid pHL73 (Table 2), thisregion was amplified under the following mutagenic condi-tions: 20 ng of supercoiled plasmid template, 1¥ AmpliTaqPCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl) (Perkin-Elmer), 0.5 mM MnCl2, 3 mM MgCl2, 0.01% gelatin, 0.3 mMof each primer, 0.2 mM of either dATP or dGTP and 1 mM ofthe other three deoxynucleotides, 5 units of AmpliTaq DNApolymerase (Perkin-Elmer) in a 100 ml PCR reaction mixture(He et al., 1996). Each reaction was preheated to 94∞C for2 min for an initial denaturation step, followed by 35 cycles(94∞C-1 min, 45∞C-1 min, 72∞C-1min) of amplification. Thereaction mixture was then incubated at 72∞C for five moreminutes to allow the completion of the partial products.The primers used for mutagenic PCR were: (i) 5¢-LexA-DB(5¢-AGGTCGTTGTCGCACGTATTGATGAC-3¢), located 152bases upstream of EcoRI site in the coding region of the lexADNA binding domain; and (ii) 3¢-AdhT (5¢-GGTAGAGGTGTG-GTCAATAAGAGCGA-3¢), located 174 bases downstream ofBamHI site in the ADH terminator sequence. A total of 500 ngof the resulting 1131 bp PCR product was co-transformedwith 50 ng of gel-purified, gapped pHL73 (generated bydigestion of pHL73 with EcoRI and BamHI to delete the eaecoding region) into L40 cells that had been pretransformedwith pHL75 (expressing Gal4(AD)-TirM128 fusion protein;Table 2). In vivo repair of the gapped plasmid with themutagenized PCR product (Muhlrad et al., 1992) resulted ina library of pHL73 derivatives, selected on SD-Trp-Leu plates,containing mutated LexA(DB)–Int181 sequences.

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Identification of LexA(BD)–Int181 mutants diminished for Gal4(AD)–TirM binding

The above transformants were replica-plated onto SD-Trp-Leu plates with 80 mg ml-1 of Xgal to screen for the white orlight blue colonies, suggesting a loss of interaction betweenLexA(DB)–Int181 and Gal4(AD)–TirM128. Cell extracts ofsuch colonies were first subjected to immunoblot analysis byusing the anti-LexA mAb (Clontech) to eliminate transfor-

mants that failed to produce the full-length LexA(DB)–Int181fusion protein. Briefly, a single yeast colony was inoculatedinto 2 ml of SD-Trp-Leu broth and grown overnight at 30∞Cwith shaking. The cells were harvested by centrifugation at2500 g for 5 min. The cell pellet was then resuspended in100 ml 1¥ SDS loading buffer (17.5 mM Tris-HCl, pH 6.8,1.75% SDS, 1% 2-mercaptoethanol, 0.01% Bromophenolblue) and incubated at 100∞C for 10 min to lyse the cells.

Plasmids from the selected yeast transformants were then

Table 2. Plasmids used in this study.

Plasmids Description Source/reference

pUCAT pUC19 derivative carrying CamR Kislauskis and Dobner (1990)pBC(KS) Vector, CamR Stratagene

Plasmids carrying full-length eaepHL6 eae in pUC19, AmpR Liu et al. (1999b)pHL109 Identical to pHL6, but with BamHI site 3¢ to eae This studypHL112 eae inserted into pBC(KS) at HindIII–BamHI sites This study

Yeast two-hybrid plasmidspBTM116 LexA fusion vector, TRP1, AmpR Bartel and FieldspGAD424 Gal4 activation domain vector, LEU2, AmpR GenBank #U07647pHF1186 pBTM116 nmd2 – produces LexA-NMD2 He et al. (1997)pHL59 pBTM116 eae (1618–2802) – produces LexA–Int395 This studypHL72 pBTM116 eae (1618–2802) – with a mutation at amino acid 932 from Cys to Ser –

produces LexA-Int395C932SThis study

pHL73 pBTM116 eae (2260–2802) – produces LexA–Int181 This studypHL74 pBTM116 eae (2503–2802) – produces LexA–Int100 This studypHF1414 pGAD424 upf3 – produces GAD–UPF3 He et al. (1997)pHL60 pGAD424 tir (1–1674) – produces GAD–TirFL This studypHL61 pGAD424 tir (736–1674) – produces GAD–TirM + C This studypHL75 pGAD424 tir (736–1119) – produces GAD–TirM128 This studypHL88 pGAD424 tir (1–711) – produces GAD–TirN This studypHL102 pGAD424 tir (1096–1674) – produces GAD–TirC This study

GST–Tir fusion plasmidspGex-2T Produces GST PharmaciapGST-TirM128 pGex-2T tir (736–1119) – produces GST-TirM128 This studypGST-Tir313 pGex-2T tir (736–1708) – produces GST-TirM313 Liu et al. (1999)pGST-TirN pGex-2T tir (1–711) – produces GST-TirN This studypGST-TirC pGex-2T tir (1096–1674) – produces GST-TirC This study

MBP–intimin fusion plasmidspMalc2 Produces MBP-b-gal a-fragment New Eng. BiolabspMal-Int395 pMalc2 eae (1618–2802) – produces MBP-Int395 Liu et al. (1999)pMal-Int395C-S pMalc2 eae (1618–2802) – produces MBP-Int395C-S, with a Cys to Ser substitution

at amino acid 932This study

pMal-Int277 pMalc2 eae (1972–2802) – produces MBP-Int277 Liu et al. (1999)pMal-Int181 pMalc2 eae (2260–2802) – produces MBP-Int181 Liu et al. (1999)pMal-Int181V-A pMalc2 eae (2260–2802) – produces MBP-Int181, with a Val to Ala substitution at

amino acid 906Liu et al. (1999)

pMal-Int100 pMalc2 eae (2503–2802) – produces MBP-Int100 Liu et al. (1999)

Alanine substitution plasmids (derived from pMal–Int277)pMal-IntS886A pMalc2 eae (1972–2802) – produces MBP-Int277, with a Ser to Ala substitution at

amino acid 886This study

pMal-IntS890A pMalc2 eae (1972–2802) – produces MBP-Int277, with a Ser to Ala substitution atamino acid 890

This study

pMal-IntT892A pMalc2 eae (1972–2802) – produces MBP-Int277, with a Thr to Ala substitution atamino acid 892

This study

pMal-IntQ897A pMalc2 eae (1972–2802) – produces MBP-Int277, with a Gln to Ala substitution atamino acid 897

This study

pMal-IntT909A pMalc2 eae (1972–2802) – produces MBP-Int277, with a Thr to Ala substitution atamino acid 909

This study

pMal-IntN916A pMalc2 eae (1972–2802) – produces MBP-Int277, with a Asn to Ala substitution atamino acid 916

This study

pMal-IntN927A pMalc2 eae (1972–2802) – produces MBP-Int277, with a Asn to Ala substitution atamino acid 927

This study

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purified from 4 ml cultures grown at 30∞C overnight in SD-Trp-Leu broth with shaking. After centrifugation at 1225 g for5 min, the cell pellet was washed once in 500 ml of steriledistilled H2O. Then, 200 ml of buffer A (2% Triton X-100, 1%SDS, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA),200 ml of chloroform/phenol, and 0.3 mg of glass beads(Sigma Chemical) were added, and the mixture was vortexedvigorously at high speed for 5 min. The supernatant wascollected after centrifugation at 16 000 g for 10 min, and thepellet was extracted again with 200 ml of Buffer A asdescribed above. DNA from the pooled supernatants wasethanol precipitated (0.3 M NaOAc, 2.5 volumes of ethanol,-20∞C, 30 min), washed once in 70% ethanol and trans-formed into E. coli K-12 strain JM109. Colonies carryingpHL73 derivatives were identified by colony PCR using the

eae-derived primer pair F-eaeC181 and R-eae (Table 3).pHL73 derivatives were isolated from JM109 and co-transformed with pHL75 into L40. The b-galactosidase activ-ity of the co-transformants was determined by ONPG liquidassays to quantitate the GAD(AD)-Tir-binding defect of themutants. The selected pHL73 derivatives were sequenced tocharacterize the mutation(s).

Generating point mutations in full-length intimin

Full-length eae derivatives carrying the mutations identifiedby the above two-hybrid analysis were cloned into high copynumber plasmids. To facilitate the transfer of eae mutationsidentified in the yeast two-hybrid plasmid pHL73 to full-length

Primera Sequenceb Positionc

LexA–intimin fusionsF-eaeC395 5¢-cgg aat tca aca atg tac agc tta cta t-3¢ 1618–1637F-eaeC181 5¢-cgg aat tcg atg aac tga aaa ttg aca a-3¢ 2260–2279F-eaeC100 5¢-cgg aat tca cta taa aag cac cgt cgt a-3¢ 2503–2522R-eae 5¢-cgg gat ccc ata gtt gtt gct tga tgt g-3¢ 2992–3011R-eaeC932S 5¢-cgg gat cct tat tct aca gaa acc gca t-3¢ 2783–2802

Gal4 AD–Tir fusionsF-tirFL 5¢-gga aga tct caa tgc cta ttg gta atc ttg g-3¢ -2 to 20F-tirM + C 5¢-cgg aat tcg cgg cga cgg gta ttg tac a-3¢ 736–755F-tirM128 5¢-cgg aat tcg cgg cga cgg gta ttg tac a-3¢ 736–755F-tirN 5¢-gga aga tct caa tgc cta ttg gta atc ttg g-3¢ -2 to 20F-tirC 5¢-cgg aat tca gtg gcg cat tga ttc ttg g-3¢ 1096–1115R-tir 5¢-aac tgc agc tcc ctc taa ata aaa tga t-3¢ 1689–1708R-tirM128 5¢-aac tgc agt taa cca aga atc aat gcg c-3¢ 1100–1119R-tirN 5¢-aac tgc agt taa gtc ccc aac gcc aac c-3¢ 695–714

GST–Tir fusionsF-tirM128-GST 5¢-gaa gat ctg cgg cga cgg gta ttg tac a-3¢ 736–755F-tirN-GST 5¢-gga aga tct atg cct att ggt aat ctt gg-3¢ 1–20F-tirC-GST 5¢-gga aga tct agt ggc gca ttg att ctt gg-3¢ 1096–1115R-tirM128-GST 5¢-ccg caa ttg tta acc aag aat caa tgc gc-3¢ 1100–1119R-tirN-GST 5¢-ccg caa ttg tta agt ccc caa cgc caa cc-3¢ 695–714R-tir-GST 5¢-ccg caa ttg ctc cct cta aat aaa atg at-3¢ 1689–1708F-tirM1-GST 5¢-gaa gat ctg cgg cga cgg gta ttg tac a-3¢ 736–755F-tirM2-GST 5¢-gga aga tct gct gca agt gca act gaa act g-3¢ 811–832F-tirM3-GST 5¢-gga aga tct ggg gta ttg aaa gat gat gt-3¢ 922–941R-tirM1-GST 5¢-ccg caa ttg tta caa tac ccc tga cgg aat c-3¢ 912–930R-tirM2-GST 5¢-ccg caa ttg ttc aat ggc ttg ctg ttt ggc-3¢ 985–1005R-tirM3-GST 5¢-ccg caa ttg tta acc aag aat caa tgc gc-3¢ 1100–1119

MBP–intimin fusionsF-eaeC395-MBP 5¢-cg gga tcc aac aat gta cag ctt act at-3¢ 1618–1637R-eaeC395C-S-MBP 5¢-caa aag ctt tta ttc tac aga aac cgc at-3¢ 2783–2802

eae and eae mutantsF-eae, 5¢-caa aag ctt cat tct aac tca ttg tgg tg-3¢ -30 to -11F-eaeC181 (PshAI)d 5¢-gcg act gag gtc act ttt ttt gat gaa ctg aaa att

gac aa-3¢2239–2279

R-eae2440 5¢-tta cga cac tgc ctt tac cat tca a-3¢ 2240–2264R-eaeC932S 5¢-tat acc cgg c tta ttc tac aga aac cgc at-3¢ 2786–2805

a. F, forward (top-strand) primer; R, reverse (bottom-strand) primer. ‘N’ or ‘C’ in primer nameindicates that the primer was used to generate an N- or C-terminal fragments, respectively. ‘M’indicates that the primer was used to generate an internal fragment. Number in primer nameindicates the number of residues of the designated protein in the final fusion product.b. Underlined sequences represent the restriction enzyme sites used for cloning.c. Numbers indicate co-ordinates of primer, with position +1 representing the first nucleotideof the coding sequence of eae or tir.d. This primer starts 21 nucleotides upstream of the 5¢ end of the Int181 coding region. Withinthese 21 nucleotides, there is a PshAI site (underlined sequence) which facilitates the transferof point mutations from pHL73 into full-length eae (see Experimental procedures).

Table 3. DNA sequences of primers used inthis study.

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eae, three pUC19-derived plasmids carrying wild-type eae,which differ only in flanking restriction sites or the antibioticmarker were generated: (i) pHL109, which carries the eaegene amplified from genomic DNA of EHEC O157:H7 strainEDL933 by the Pfu Turbo PCR system (Stratagene) usingprimers F-eae, and R-eae (Table 3), inserted into the HindIIIand BamHI sites of pUC19; (ii) the previously describedpHL6 (Liu et al., 1999), which is identical to pHL109 exceptthat it lacks the downstream BamHI site; and (iii) pHL112,generated by subcloning the eae-containing HindIII–BamHIfragment from pHL109 into pBC(KS) (Stratagene), achloramphenicol-resistant derivative of pUC19.

The eae mutations in pHL73 derivatives identified by thetwo-hybrid screen were transferred to pHL109, pHL6 orpHL112 by two different methods, depending on the locationof the mutations. G781D and W796R, two mutations located5¢ to the NsiI site at position 2420–2426, were amplified fromthe pHL73 derivatives by Pfu (turbo) PCR system using F-eaeC181(PshAI) and R-eae2440 (Table 3), resulting in aneae gene fragment carrying the mutation and flanked byPshAI and NsiI restriction sites. The equivalent PshAI–NsiIfragment from pHL6 was replaced with this amplified product,resulting in pUC19-derived plasmids encoding full-lengthintimin carrying either G781D or W796R. All mutations wereconfirmed by DNA sequencing.

eae mutations identified in the two-hybrid screen that werelocated 3¢ of the NsiI site were moved to full length eae byreplacing the Nsi I–BamHI restriction fragment of pHL109,pHL112 (or both) with the equivalent fragment from thepHL73 derivatives. The mutations V845A, P863L, Y882C,Q902L, G905E, V906E, N927D, V928I, V931D were movedinto pHL109, and the mutations S826P, P839L, W894R,N911I were moved into pHL112. In all cases, the mutationwas confirmed by DNA sequencing. (Three mutations,P839L, Y882C, and N911I, were moved into both pHL109and pHL112, and the same mutation in either plasmid gaveroughly equivalent results for GST-Tir binding and inductionof AE lesions; data not shown. In Table 1, data shown are forY882C expressed from pHL109, and for P839L and N911Iexpressed from pHL112.)

Site-directed mutagenesis

To generate a Cys to Ser mutation at intimin residue 932, thecoding region of the intimin C-terminal 181 amino acids wasamplified from EDL933 genomic DNA by the high-fidelityPCR system (Boehringer Mannheim) using the primer pairsF-eaeC181 and R-eaeC932S, which was designed to givethe Cys to Ser mutation at amino acid 932 (Table 3). The PCRproduct was cleaved with NsiI and XmaI, and the resulting250 bp fragment was used to replace the correspondingNsiI–XmaI fragment of plasmid pHL6, a pUC19-derived plas-mid carrying wild-type eae (Table 2). The identical mutationwas generated in pMal-Int395, which encodes an MBP–intimin fusion protein carrying the C-terminal 396 residuesof intimin, using an equivalent strategy (see primers F-eaeC395-MBP and R-eaeC395C-S-MBP; Table 3).

Point mutants in the MBP-Int277 fusion protein were gen-erated using the QuikChange Site-Directed Mutagenesis Kit(Stratagene) according to the manufacturer’s instructions.Oligonucleotides (Life Technologies) were high-performanceliquid chromatography (HPLC)-purified and are listed inTable 4. All mutations were confirmed by DNA sequencing.

Expression of intimin mutants on the bacterial surface

The high-copy number plasmids carrying the above eaemutations were transformed into an E. coli K-12 strainMC1061. The level of expression of the intimin mutants onthe bacterial surface was determined by enzyme-linkedimmunoabsorbance assay (ELISA), as described previously(Liu et al., 1999). Briefly, 1.8 ¥ 107 washed bacteria in PBSimmobilized in poly L-lysine-treated 96-well microtitre wells(Product #3072; Becton-Dickenson Labware) and then fixedin 3% paraformaldehyde in phosphate-buffered saline (PBS)for 1 h at 20∞C. After blocking for 1 h at 20∞C with PBS/5%milk, the wells were probed with sheep anti-EHEC intiminserum (raised against the C-terminal 300 amino acids ofintimin from EHEC strain 86–24; gift of Alison O’Brien) diluted1:500, and then horseradish peroxidase-conjugated anti-sheep IgG (Sigma). The colorimetric peroxidase substrate

Primera Sequenceb Positionc

Intimin mutantsS886A 5¢-gcc att atg ctt cta tga act caa taa ctg ctt gg-3¢ 2648–2682S890A 5¢-gcc att ata gtt cta tga acg caa taa ctg ctt gg-3¢ 2648–2682T892A 5¢-gaa ctc aat agc tgc ttg gat taa aca gac atc tag-3¢ 2664–2699S899A 5¢-gga tta aac aga cag cta gtg agc agc gtt ctg g-3¢ 2681–2714S900A 5¢-gga tta aac aga cat ctg ctg agc agc gtt ctg g-3¢ 2681–2714E901A 5¢-cag aca tct agt gcg cag cgt tct gga gta tc-3¢ 2689–2720N911A 5¢-gga gta tca agc act tat gcc cta ata aca caa aac c-3¢ 2713–2749T914A 5¢-ctt ata acc taa tag cac aaa acc ctc ttc ctg ggg-3¢ 2726–2761N927A 5¢-ggt taa tgt taa tac tcc agc tgt cta tgc gg-3¢ 2760–2791Q897A 5¢-gct tgg att aaa gcg aca tct agt gag cag-3¢ 2677–2706T909A 5¢-gcg ttc tgg agt atc aag cgc tta taa cct aat aac-3¢ 2706–2741N916A 5¢-act tat aac cta ata aca caa gcc cct ctt cct g-3¢ 2725–2758

a. Letter and number in primer name indicate the residue that was altered to alanine.b. Only the sequence of the top-strand primer is shown; bottom-strand primers consisted ofthe complementary sequence. Underlined bases were mutated from wild type to encodealanine residue.c. Numbers correspond to the position of the primer in the eae coding sequence, with the ATGstart codon as positions +1 to +3.

Table 4. DNA sequences of primers used foralanine substitutions.

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(TMB Microwell) was added to quantitate bound anti-intiminantibody (Sigma 104, Sigma Chemical Co). To normalize forvariations in the number of bacteria, the same wells weresubjected to ELISA using rabbit anti-OmpA antiserum (gift ofC. Kumamoto) diluted 1:5000 and HRP-conjugated goat anti-rabbit IgG. The sheep anti-intimin ELISA signal was dividedby the anti-OmpA ELISA signal to give an intimin:OmpAexpression ratio, and the expression ratio for each intiminderivatives was corrected for non-specific binding by sub-tracting the background ELISA signal of sheep anti-intiminbinding to MC1061 harbouring the negative control plasmid,pUCAT (Kislauskis and Dobner, 1990). The resulting expres-sion index of MC1061 harbouring wild-type intimin wasassigned a relative value of 1.00, and the expression of eachof the intimin mutants was expressed relative to this value.We found significant experiment-to-experiment variation inthe surface expression of intimin, and therefore testedmutants as many as seven times. Values shown in Table 1are from a representative experiment, and are neither thehighest nor the lowest values obtained for that mutant uponrepeated determination.

Tir-binding by bacteria expressing intimin mutants

In experiments performed in parallel to those describedabove, microtitre wells containing bacteria were probed with2 mg ml-1 of GST–Tir313 or, as a control, GST. Bound GST-Tir (or GST) was quantitated by ELISA using goat anti-GSTantibody (Pharmacia Biotech) diluted 1:1000 followed byalkaline phosphatase-conjugated anti-goat IgG (SigmaChemical Co.) diluted 1:10 000 and colorimetric alkalinephosphatase substrate, as described previously (Liu et al.,1999). To normalize for variations in cell number, identicallytreated wells were subjected to ELISA in parallel using rabbitanti-OmpA antiserum (a gift of Carol Kumamoto) diluted1:5000 and HRP-conjugated goat anti-rabbit antibody. Foursteps of correction and normalization were performed toobtain a Tir-specific binding signal. First, the anti-GST-TirELISA signal for each bacterial strain was divided by the anti-OmpA ELISA signal to give a GST-Tir:OmpA ratio (or, whenGST alone was used as a probe, a GST:OmpA ratio). Sec-ond, the GST:OmpA ratio was subtracted from the GST-Tir:OmpA ratio. Third, this normalized GST-Tir:OmpA ratiowas further corrected for non-specific binding by subtractingfrom it the (background) GST-Tir:OmpA ratio obtained forMC1061 harbouring the pUCAT vector alone (Kislauskis andDobner, 1990). Finally, the Tir-specific binding value of eachmutant was expressed relative to that of MC1061 harbouringpHL6, which encodes wild-type intimin and was assigned arelative value of 1.00.

Ability of intimin mutants to bind to or trigger actin condensation in preinfected mammalian cells

The ability of E. coli MC1061 expressing the intimin mutantsto trigger actin condensation in HEp-2 cells preinfected withan EPEC eae mutant was assessed as described previously(Liu et al., 1999). Briefly, monolayers of HEp-2 cells werepreinfected with the EPEC eae mutant JPN15.96/pMAR7 for3 h at 37∞C, then treated with gentamicin. Overnight culturesof MC1061 expressing intimin were added to preinfectedmonolayers and incubated for 3 h at 37∞C. After washing,

fixing, and permeabilizing the monolayers, the cells wereincubated with 5 mg ml-1 of TRITC-phalloidin and examinedby phase contrast and fluorescent microscopy (Knutton et al.,1989).

To test for the ability of E. coli K12 expressing intimin orintimin mutants to bind to Tir on the surface of mammaliancells, HEp-2 cells were mock infected or preinfected withJPN15.96/pMAR7 at a multiplicity of infection (moi) of 200,then gentamicin treated as described above. Monolayerswere then infected at an moi of approximately 10 with E. coliMC1061 expressing intimin or intimin mutants and, after90 min at 37∞C, were washed six times with PBS to removeunbound bacteria. Stably bound bacteria were quantitated byplating for viable counts on Luria–Bertani (LB) agar withappropriate antibiotic after dissociation of bacteria and HEp-2 cells with 0.5% Triton X-100.

Gel overlay assays

The MBP–intimin and GST–Tir fusion proteins were purifiedas described previously (Liu et al., 1999). The concentrationof purified proteins were estimated using the BCA proteindetermination kit (Pierce Chemical) and by visual comparisonof band intensity after Coomassie blue staining of SDS poly-acrylamide gels containing dilutions of purified proteins andknown amounts of marker proteins. (The latter method wasrequired when trying to estimate the concentration of full-length fusion protein when the sample contained degradationspecies.)

MBP-intimin derivatives were separated by SDS-PAGE,transferred onto a nitrocellulose membrane and blockedwith PBS + 5% milk for 1 h at room temperature (RT). Themembrane was then probed with 2 mg ml-1 of GST-TirM inPBS + 0.2% BSA for 3 h at RT or at 4∞C overnight, andblocked again in PBS + 5% milk for 1 h. The GST–TirMbound to the membrane was detected by goat anti-GSTantiserum (1:1000, Pharmacia Biotech) and rabbit anti-goatIgG conjugated to alkaline phosphatase (Sigma ChemicalCo.).

Modified ELISA to detect the interactions between MBP–intimin and GST–Tir fusions

To obtain quantitative measures of the binding activity ofMBP–intimin fusion proteins to GST–Tir derivatives, 96-wellLinbro microtitre plates (ICN/Flow Biomedicals) were coatedwith MBP–intimin fusion proteins at 4 mg ml-1 in coating buffer(50 mM Na2CO3, 50 mM NaHCO3 pH 9.6) at 4∞C overnight.Wells were then blocked in PBS/3.5% BSA for 2 h at 20∞Cand probed with 2 mg ml-1 of GST-TirM in PBS + 0.2% BSAfor 3 h at 20∞C. Wells were washed and fixed overnight in 3%paraformaldehyde in PBS and blocked again in PBS/5%skimmed milk. Bound GST–Tir was revealed by goatanti-GST antiserum (1:1000, Pharmacia Biotech) and rabbitanti-goat IgG conjugated to alkaline phosphatase (SigmaChemical Co.).

Interpreting the EHEC sequence based on the EPEC intimin–Tir complex crystal structure

The complex of EHEC intimin and Tir was modelled basedon the crystal structure of the EPEC intimin and Tir (Luo

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© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573

et al., 2000). The proteins from the two species are homolo-gous with amino acid sequence alignment resulting in 47%sequence identity for the intimin and 72% for Tir. The complexof EHEC was mapped onto the EPEC crystal structure(1F00) using the interactive graphics package MIDAS (Ferrinet al., 1988) to replace the homologous residues and thengenerate the figures. Note: the numbering on the figures ofintimin and Tir is that of EHEC.

Acknowledgements

Ralph Isberg provided invaluable advice and recombinantinvasin protein. We thank Zsuzsa Hamburger and PamelaBjorkman for assistance in the analysis of the structure ofinvasin, and for communication of unpublished results. PaulFurcinetti of the Digital Imaging Facility at University ofMassachusetts Medical School provided assistance in gen-erating fluorescent microscopy images. Alison O’Brien gen-erously provided anti-intimin antiserum and Carol Kumamotogenerously provided anti-OmpA antiserum. We thank JonGoguen and Donald Tipper for helpful discussion and carefulreview of the manuscript. This work was supported by NIH-R01-AI46454 and by funding from the Worcester Foundationfor Biomedical Research to J.M.L. J.M.L. is an EstablishedInvestigator of the American Heart Association. This publica-tion’s contents are solely the responsibility of the authors anddo not necessarily represent the official views of the Worces-ter Foundation, the NIH or the American Heart Association.

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