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Molecular Microbiology (2002) 45(6), 1557–1573
© 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. Schiffer2 and
John M. Leong1*
Departments of 1Molecular Genetics and Microbiology,
and 2Biochemistry and Molecular Pharmacology,
University of Massachusetts Medical School, Worcester,
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 and
effacing (AE) lesion. Intimin, an outer membrane
protein of EHEC, is required for the formation of AE
lesions, as is Tir, a bacterial protein that is translo-
cated into the host cell to function as a receptor for
intimin. We established a yeast two-hybrid assay for
intimin–Tir interaction and, after random mutagene-
sis, isolated 24 point mutants in intimin, which
disrupted Tir recognition in this system. Analysis of
11 point mutants revealed a correlation between rec-
ognition of recombinant Tir and the ability to trigger
AE lesions. Many of the mutations fell within a 50-
residue region near the C-terminus of intimin.
Alanine-scanning mutagenesis of this region revealed
four residues (Ser890, Thr909, Asn916 and Asn927)
that are critical for Tir recognition. Mapping the
sequences of EHEC intimin and Tir onto the crystal
structure of the intimin–Tir complex of enteropatho-
genic E. coli predicts that each of these four intimin
residues 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 both
identified residues critical for Tir binding and demon-
strated a correlation between the ability to bind Tir
and the ability to trigger actin focusing.
Enterohaemorrhagic Escherichia coli (EHEC) has
emerged as one of the most important food-borne patho-
gens in North America, Europe and Japan (reviewed in
Nataro and Kaper, 1998). It is responsible for both
sporadic cases and epidemic outbreaks of haemorrhagic
colitis, and is associated with the haemolytic uraemic syn-
drome (HUS), the triad of haemolytic anaemia, thromb-
ocytopenia and renal failure. This life-threatening
complication is due to the high level expression of shiga-
like toxins (Stx's; O'Brien et al., 1992) by bacteria that
remain bound to the intestinal mucosa.
Colonization of the intestinal mucosa by EHEC, as well
as by a subset of other intestinal pathogens such as
enteropathogenic E. coli (EPEC), involves the formation
of 'attaching and effacing (AE) lesions'. Epithelial cell
microvilli are effaced and bacteria are closely opposed to
the host cell membrane. Most strikingly, filamentous actin
is generated beneath the attached bacteria, resulting in
the formation of 'pedestals' that lift the bacteria above the
plane of the host cell membrane (Staley et al., 1969;
Ulshen and Rollo, 1980; Moon et al., 1983). A contiguous
35 kb pathogenicity island, termed the locus of enterocyte
effacement (LEE) is associated with the ability to form AE
lesions (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 as
the bacterial effector molecules that are translocated by
this apparatus to the host cell (Jarvis et al., 1995; Kenny
and Finlay, 1995).
A multistep model has been proposed for AE lesion
formation (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 in
effacement of microvilli (Donnenberg and Kaper, 1991;
Foubister et al., 1994; Ismaili et al., 1995; Kenny and
Finlay, 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 localizes
in the host cell membrane. Remarkably, Tir acts as a
receptor for the bacterial outer membrane protein intimin,
encoded by the eae gene (Jerse, 1990; Kenny et al.,
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1558 H. Liu et al.
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1997). The interaction of Tir and intimin triggers the final
step of AE lesion formation, leading to actin condensation,
pedestal formation and intimate bacterial attachment.
Consistent with the hypothesis that this process can occur
in discrete steps, laboratory strains of E. coli expressing
intimin, or even intimin-coated latex beads, form AE
lesions on mammalian cells that have been preinfected
with 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 intestinal
colonization in experimental animals (Donnenberg et al.,
1993; Marches et al., 2000) as well as AE lesion formation
indicates an important role for intimate attachment during
Several recent studies have shed light on the structure
and function of intimin. The crystal structure of the C-
terminal extracellular domain of the intimin-related Yers-
inia pseudotuberculosis invasin protein revealed an
elongated 180 Å rod consisting of four domains resem-
bling immunoglobulin superfamily (IgSF) domains and a
fifth domain related to C-type lectin-like domains
(Hamburger et al., 1999). The last IgSF and the C-type
lectin-like domains form a superdomain that is sufficient
to bind host cell invasin receptors (a subset of b1-chain
integrins) (Leong et al., 1990; Rankin et al., 1992).
Despite limited sequence identity between invasin and
intimin, the global fold of the last 280 amino acids (i.e. last
three domains) of EPEC intimin, determined by multidi-
mensional magnetic resonance (Kelly et al., 1999), also
consists of two IgSF domains and a C-type lectin-like
domain, and the last two domains were necessary and
sufficient for receptor (i.e. Tir) binding (Liu et al., 1999).
This minimal Tir-binding region of intimin, when purified
and immobilized on latex beads, was also sufficient to
trigger AE lesions on mammalian cells that had been
preinfected with E. coli competent for type III secretion
(Liu et al., 1999). Nuclear magnetic resonance (NMR)
titration analysis of EPEC intimin and Tir implicated a
50 aa region at the C-terminus of intimin in Tir recognition
(Batchelor et al., 2000). Finally, the recently published
EPEC 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 that
disrupted Tir recognition. The ability of intimin mutants to
bind to recombinant Tir correlated with their ability to trig-
ger AE lesions on preinfected mammalian cells. Half of
the 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 intimin
that are critical for Tir recognition. In a model of the EHEC
intimin–Tir complex that is based on EPEC intimin and
Tir (Luo et al., 2000), these four amino acids are predicted
to be located at the intimin–Tir interface, indicating that
these residues play a functional role in intimin recognition
by Tir.
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 AE
lesions, we sought to isolate mutations in intimin that
diminished Tir recognition. The yeast two-hybrid system
can provide a simple screen for mutations that diminish
protein–protein interactions, so we first optimized a two-
hybrid assay for intimin-Tir binding. Full-length Tir was
fused 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 domain
of intimin (Hamburger et al., 1999; Batchelor et al., 2000;
Luo et al., 2000), were fused to the LexA DNA-binding
domain in a yeast two-hybrid vector to generate fusion
protein LexA–Int395 (Fig. 1A; see Experimental proce-
dures). Yeast transformants expressing LexA–Int395 and
Gal4 AD–Tir produced ª 27 Miller units of b-galactosidase
activity, i.e. approximately 10-fold greater than the
background levels determined by control transformants
expressing a LexA fusion to the yeast protein NMD2 (He
et al., 1996) and Gal 4 AD–Tir (Fig. 1A). The C-terminal
181 aa of intimin are sufficient to bind recombinant Tir
(Liu et al., 1999; Batchelor et al., 2000), and a yeast strain
expressing LexA–Int181 and Gal4 AD–Tir produced
higher levels of b-galactosidase activity (ª 400 Miller
units). The C-terminal 100 residues of intimin are devoid
of Tir binding activity, and as expected, coexpression
of LexA–Int100 with Gal4AD–Tir did not result in b-
galactosidase activity above background levels. The two
transmembrane domains of Tir (Kenny et al., 1997) divide
this protein into three intervals, an N-terminal (TirN), a
central (TirM) and C-terminal region (TirC) (Fig. 1B). The
intimin-binding region of EPEC Tir has been mapped to
the central domain, TirM (de Grado et al., 1999; Hartland
et al., 1999; Kenny, 1999). To optimize signal strength in
the two-hybrid assay, GAD–Tir fusion proteins carrying
one or more of these Tir domains were generated and
coexpressed 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 of
LexA–Int181 and the Gal4 AD fused to the yeast protein
UPF3 (He et al., 1997; Fig. 1B). Yeast transformants coex-
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Point mutants of EHEC intimin that diminish Tir binding1559
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573
pressing Gal4 AD fusions carrying the central region of
Tir (TirN + M + C, TirM + C, or TirM) and LexA–Int181
resulted in b-galactosidase activity at least 100-fold above
background levels. The smallest fusion, Gal4 AD–TirM,
gave the highest b-galactosidase activity, giving rise to
dark blue colonies on Xgal plates and almost 2000 Miller
units, some 600-fold higher than background levels. The
intimin mutant V906A diminishes Tir binding (Liu et al.,
1999), and when this mutation was generated in
LexA–Int181, the two-hybrid signal was diminished
approximately fourfold (Fig. 1C). Yeast transformants
expressing 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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© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573
Tir binding domain, produced only background levels of
b-galactosidase activity when coexpressed with Gal4
AD–TirM (Fig. 1C).
To confirm that the transcriptional activation detected in
the yeast two-hybrid system reflected a direct interaction
between EHEC intimin and Tir, various MBP–intimin
fusion proteins were tested for binding to purified GST–
TirN, GST–TirM or GST–TirC in gel overlay assays. Only
GST–TirM demonstrated intimin-binding activity (Fig. 1D
and data not shown), and this binding, as predicted,
required the C-terminal 181 aa of intimin, because fusion
protein MBP-Int100 carrying only the last 100 aa of intimin
showed no binding activity (Fig. 1D, 'Int100'). Disruption
of 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 mutation
previously shown to disrupt Tir binding (Liu et al., 1999)
(Fig. 1D, 'Int181V-A'). These data indicate that the central
region of EHEC Tir directly interacts with intimin, in parallel
with Tir–intimin interactions of EPEC (de Grado et al.,
1999; Hartland et al., 1999; Kenny, 1999), and suggest
that 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 above
to identify intimin mutants that were diminished in Tir-
binding. Although coexpression of LexA–Int181 (from
pLexA–Int181) and Gal4 AD–TirM resulted in dark blue
colonies on Xgal plates, about 500 white or light blue
colonies were identified upon screening approximately
25 000 yeast colonies after random mutagenesis of the
intimin coding region of pLexA–Int181 (see Experimental
procedures). Whole-cell extracts from these selected
yeast colonies were immunoblotted using an anti-LexA
mAb to identify strains that express full-length LexA–
Int181 fusion protein. A total of 24 plasmids that express
full-length fusion protein were characterized by sequenc-
ing their respective intimin coding regions, revealing 16
single-site mutants, six double-site mutants and two triple-
site mutants (Fig. 2). Plasmids encoding most of these
LexA–Int181 point mutants were retransformed into a
yeast 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.
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Point mutants of EHEC intimin that diminish Tir binding1561
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573
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 the
mutations altered residues that were completely con-
served among the intimin sequences from EHEC
O157:H7, EPEC E2348/69, and Citrobacter rodentium.
Given that these sequences share only 36% identity over
the 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 when
the protein was expressed at relatively high levels on the
surface of E. coli, we generated 14 of the single-site
mutations in full-length eae on a high copy number plas-
mid (see Experimental procedures). MBP-Int395 carrying
the mutation C932S lacks the disulphide loop at intimin
C-terminus (Kelly et al., 1999; Luo et al., 2000) and is
defective at binding GST–TirM (Fig. 1D). Full-length
intimin with the identical mutation was generated and
included in this analysis as a binding-incompetent control.
Pilot experiments revealed that expression of intimin from
high-copy number plasmid in the EHEC strain EDL933 did
not result in intimin easily detectable on the bacterial
surface (data not shown). Therefore, the intimin mutants
were 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, by
probing intact, immobilized bacteria with anti-intimin anti-
serum in a modified enzyme-linked immunoabsorbance
assay (ELISA) (Liu et al., 1999; see Experimental proce-
dures). Four of the mutants gave ELISA signals at levels
at least fivefold lower than the wild type (Table 1).
Although we cannot rule out the possibility that the
diminished ELISA signal of these mutants is due to dimin-
ished antibody affinity, the simplest interpretation, given
the polyclonal nature of the antiserum, is that these muta-
tions disrupt the folding and/or secretion of full-length
intimin. 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 required
for intimin function (Batchelor et al., 2000). The other 11
intimin 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 also
observed when full-length intimin was expressed on the
bacterial surface, the ability of bacteria expressing these
mutants to bind to GST-Tir was determined (see Experi-
mental procedures). As expected, intimin mutants that
were poorly expressed on the bacterial surface did not
bind to GST–Tir significantly above background levels
(Table 1). Mutants that were expressed on the bacterial
surface demonstrated a wide range of GST-Tir binding
activity. Three mutations, including the C932S mutation
that disrupts the C-terminal disulphide loop, abolished
GST–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 EPEC
intimin function (Reece et al., 2001). Three mutants,
Q902L, V845A, and V931D, bound GST-Tir at near
(≥88%) wild-type levels. The remaining five mutants
bound GST–Tir between 45% and 83% of wild-type levels.
Thus, although all of the intimin mutations appeared to
disrupt Tir recognition in the yeast two-hybrid assay, most
of the mutations did not severely diminish binding of
GST–Tir by bacteria expressing relatively high levels of
full-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 essential
activity of intimin in triggering the final step of AE lesion
formation, we tested whether these intimin mutants could
trigger the last step of AE lesion formation. E. coli K12
expressing EHEC intimin from a high-copy number plas-
mid is able to generate robust AE lesions on mammalian
cells preinfected with JPN15.96, an EPEC eae mutant that
retains the ability to secrete effector molecules (including
Tir) via the type III machinery (Jerse, 1990). This obser-
vation suggested that the Tir-binding domain of EHEC
intimin, 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 is
supported by direct binding studies (DeVinney et al.,
1999). (EHEC form AE lesions in vitro much less effi-
ciently than EPEC (Cantey and Moseley, 1991), and
preinfection of monolayers with EHEC did not result in
AE lesions upon subsequent challenge with E. coli K12
expressing intimin; data not shown).
We assessed both the quantity of AE lesions generated
by the mutants (by counting the number of AE lesions
formed per 100 mammalian cells) and the quality of those
lesions (by visually scoring the strength of phalloidin stain-
ing of F-actin) (Table 1; see Experimental procedures).
This analysis revealed a general correlation between Tir
binding activity and AE lesion formation. As expected,
mutants with virtually no GST–Tir binding activity did not
trigger AE lesions (Table 1) and, in fact, were not able to
bind efficiently to the preinfected monolayers (Fig. 3).
Mutants that retained the ability to bind GST–Tir at near
wild-type levels (Q902L, V845A and V931D) triggered AE
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© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573
lesions indistinguishable in quantity or quality from wild
type. Four out of the five remaining mutants, which were
only moderately defective for binding GST-Tir (45% to
83% wild-type levels) triggered AE lesions less robust in
phalloidin staining than wild type (Table 1, Fig. 4). Three
of these mutants also triggered significantly fewer AE
lesions, and their efficiency of AE lesion formation corre-
lated with their respective GST–Tir binding activities. One
out of the five mutants, Y882C, demonstrated a partial
defect in GST-Tir binding (63% of wild-type levels), but had
no apparent defect in AE lesion formation. One potential
explanation for this result is that the mutation may alter
intimin binding to a soluble, recombinant fragment of
EHEC Tir (GST–TirEHEC) more significantly than it does
binding 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 is
expressed at high levels on the bacterial surface can
result in significant defects in AE lesion formation. In addi-
tion, the general correlation between GST–Tir binding and
actin pedestal formation on preinfected cells suggests
that Tir binding activity is the critical property of intimin
required 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
32.6 ± 1.6
Intimin expressed poorly on the bacterial surfaceb
0.20 ± 0.06
0.00 ± 0.01
0.06 ± 0.02
0.14 ± 0.07
0.05 ± 0.02
0.00 ± 0.01
0.00 ± 0.01
-0.03 ± 0.10
Intimin expressed well on the bacterial surfaceb
0.78 ± 0.09
0.67 ± 0.06
0.89 ± 0.07
0.53 ± 0.06
0.85 ± 0.03
0.44 ± 0.04
0.98 ± 0.02
0.78 ± 0.01
0.68 ± 0.05
0.78 ± 0.07
0.85 ± 0.03
1.23 ± 0.09
0.94 ± 0.15
0.88 ± 0.05
0.69 ± 0.11
0.63 ± 0.02
0.58 ± 0.04
0.56 ± 0.01
0.45 ± 0.03
0.05 ± 0.01
0.03 ± 0.01
-0.02 ± 0.01
27.7 ± 2.2
30.5 ± 2.8
27.6 ± 2.2
23.9 ± 3.0*
29.7 ± 2.7
29.2 ± 3.5
13.8 ± 1.1*
6.4 ± 0.5*
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-intimin
antiserum, and was normalized to the level of expression of wild-type intimin (see Experimental procedures). Mutants that expressed intimin at
levels 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 are
the 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 were
normalized to the level of Tir-binding by wild-type intimin (see Experimental procedures). Shown are the mean and standard deviation of four
d. HEp-2 cells were preinfected with an enteropathogenic Escherichia coli (EPEC) eae mutant, and the indicated bacterial strain was added to
these preinfected monolayers at a multiplicity of infection (moi) of approximately 5. Each sample was coded and then scored blindly for attaching
and 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 each
experiment, mutants were tested in triplicate. Each mutant was analysed in six independent experiments, and shown are the mean and standard
error of the resulting 18 determinations. Asterisks indicate p < 0.05 for the difference between E. coli expressing mutant or wild-type intimin in
the 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 lesions
weakly stained. Each mutant was scored three times per experiment, and each experiment was performed six independent times. The consensus
score shown was obtained in at least 12 out of the 18 determinations.
g. NA, not applicable.
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Point mutants of EHEC intimin that diminish Tir binding1563
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573
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, a
residue that was implicated previously as a residue critical
for Tir binding (Liu et al., 1999). In contrast, all of the eight
mutations that fell outside of this 50-residue region were
non-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). We
therefore subjected residues in this region to alanine
scanning mutagenesis. At the time that these mutants
were generated, 33 intimin sequences had been identified
and the crystal structure of the Y. pseudotuberculosis
invasin had been published (Hamburger et al., 1999). We
substituted alanine for 10 amino acids in this interval that
fulfilled the following criteria. First, these residues were
conserved among at least 27 out of the 33 sequenced
members of the intimin family (Yu and Kaper, 1992;
Beebakhee et al., 1992; An et al., 1997). Second, these
were residues for which alanine was not found in any of
the sequenced intimin proteins. Third, to target residues
more likely to be on the surface of intimin, we did not
mutate codons specifying aliphatic or aromatic amino
acids, or residues that corresponded to buried residues in
Y. 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 encoded
MBP-Int277, a maltose binding protein fusion that con-
tains the C-terminal 277 amino acids of EHEC intimin (Liu
et 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 the
protein and subsequent degradation. The other seven
mutants demonstrated a variable degree of degradation
products after purification (Fig. 5A), but were purified in
sufficient 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.
Page 8
1564 H. Liu et al.
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573
We examined the Tir binding activity of each of the
purified mutants by a gel overlay assay, probing filters with
recombinant GST-TirM and revealing bound GST-Tir with
anti-GST antibody. At least three mutants, S890A, T909A,
and N927A, were diminished in Tir binding (Fig. 5A). To
obtain a more quantitative assessment of defects in Tir-
binding by the intimin mutants, microtitre wells coated with
wild-type or mutant MBP-intimin fusion proteins were
probed with recombinant TirM. The same three mutants
demonstrated severe (p < 0.0001) defects in Tir recogni-
tion in this assay (Fig. 5B). In addition, Tir-binding by
N916A was partially but significantly (p = 0.0001) dimin-
ished, i.e. demonstrated binding at least 21% compared
with that of wild-type MBP-intimin in repeated experiments
(Fig. 5B and data not shown).
To determine the likely location of the above residues
with respect to the intimin–Tir binding interface, we
mapped the sequences of EHEC intimin and Tir onto the
known 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 are
72% identical and the Tir binding domains of EPEC and
EHEC intimin are 47% identical, and the sequence align-
ments can be made without introducing any insertions (Yu
and Kaper, 1992; Elliott et al., 1998; Perna et al., 1998).
Thus, the resulting model in which aligned residues are
swapped should be physically reasonable. Analysis of the
alanine scanning data in terms of this model shows that
Q897A and S886A, which have no discernible binding
phenotype, are remote from the presumed binding inter-
face. T892A, which also exhibits no binding phenotype, is
in the middle of the interface. Although the alanine sub-
stitution for the larger residue threonine probably results
in a small indentation on the intimin surface, sequence
mapping analysis suggests that T892 is not in the vicinity
of potential hydrogen bonding partners and does not
make van der Waal's contact with Tir. Thus, it is reason-
able that T892A displayed no detectable Tir binding
The four residues that were identified as critical for Tir
binding, S890, T909, N916 and N927, are predicted to
form a small groove that surrounds the side chain of Tir
I298 (Fig. 6). Intimin residue T909 (along with residues
T892, V921 and V928) is at the base of the putative
binding groove, whereas S890, N916 and N927 surround
the edge. N927 is predicted to make the closest contact
with Tir, consistent with the observation that the intimin
mutant N927D that was isolated from our initial screen
was highly defective for promoting AE lesion formation
when expressed on the bacterial surface (Table 1). Thus,
the intimin residues critical for Tir binding identified by this
genetic approach are likely to directly interact with Tir, and
the location of these residues specifically highlight a puta-
tive Tir binding groove in which Tir residue I298 fits.
One of the goals of this study was to identify residues of
EHEC intimin that are critical for Tir binding. For this
purpose, we isolated mutants that were defective for
Tir-binding in a yeast two-hybrid assay after random
mutagenesis of intimin's Tir binding domain. Half of the
mutations, including the most conservative substitutions,
fell within a 50-residue C-terminal region of intimin previ-
ously implicated in Tir binding by NMR analysis (Batchelor
et 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 protein
surface. Alanine substitution of four of these residues
significantly 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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Point mutants of EHEC intimin that diminish Tir binding1565
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573
sequence of EHEC intimin and Tir onto the recently deter-
mined EPEC intimin–Tir complex crystal structure (Luo
et al., 2000), these four amino acids are likely to be a part
of a Tir-binding groove on the surface of intimin. In con-
trast, residues that did not alter Tir binding upon alanine
substitution are predicted either to not interact with Tir, or
to interact only through backbone interactions that may
not 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.
Page 10
1566 H. Liu et al.
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573
plus the fact that the Tir-binding region of intimin and the
intimin-binding region of Tir are both highly conserved
between EPEC and EHEC, provide support for the accu-
racy of the mapping of the EHEC sequence onto the
EPEC intimin–Tir complex crystal structure. Consistent
with this assertion, an alanine substitution mutant of
EPEC intimin (I897A) that is equivalent to the EHEC
intimin T892A shown here to retain Tir binding activity,
also bound Tir indistinguishably from wild-type (Reece
et al., 2001).
The ultimate identification of critical intimin residues
validates the use of the yeast two-hybrid screen for this
purpose, but this assay system did not uniformly reflect
Tir-intimin binding by other assays. For example, most of
the residues that were identified by the two-hybrid screen
(i.e. all except V906, N911 and N927) are predicted to
be far from the binding interface (Luo et al., 2000); P.
Radhakrishnan, M. Prabu and C. Schiffer, unpublished
observations), and mutation of some of these had little
effect on binding of intimin to recombinant Tir in vitro or
on triggering actin pedestals on cells preinfected with
EPEC. Furthermore, inclusion of regions of Tir or intimin
outside of their interacting domains reduced the signal in
the two-hybrid assay, suggesting that the size of the fusion
proteins and/or their ability to localize to and fold properly
in the yeast nucleus are likely to have large effects in this
assay system. Consistent with the idiosyncratic nature of
the two-hybrid assay system for Tir–intimin interactions,
two-hybrid signals for EPEC intimin and Tir are relatively
weak (de Grado et al., 1999; Hartland et al., 1999).
Although Tir recognition by EPEC and EHEC intimin is
clearly similar, some equivalent mutants in these two
proteins display apparently discrepant phenotypes.
Whereas no Tir-binding defects in EPEC intimin mutants
T914A and C937S were detected in two-hybrid or gel
overlay assays (Hartland et al., 1999; Reece et al., 2001),
the corresponding EHEC mutants T909A and C932S
described here were defective for Tir binding in gel over-
lay or modified ELISA assays, or both (Figs 1D and 5). In
addition, bacteria expressing EHEC intimin C932S bound
neither to recombinant EHEC Tir (Table 1), nor to mono-
layers expressing EPEC Tir in the eukaryotic membrane
by 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, unpublished
observations), indicating that the apparent degree of Tir
binding activity retained by an intimin mutant can vary
with the assay type and with the particular form of intimin
derivative. Supporting this notion, recombinant proteins
carrying the extracellular domain of intimin display bind-
ing activities that are not detectable for full length intimin
expressed 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 to
differences in the intimin and Tir sequences of the two
pathogens, it is possible that EPEC Int T914A and C937S
have Tir binding defects that are not detectable in some
assays, for example, two-hybrid or gel overlay assays
(Hartland et al., 1999; Reece et al., 2001). EPEC Int
C937S or IntT914A do not show the full range of biologi-
cal activity of intimin, and it has been postulated that this
failure is due to their inability to bind an unidentified
endogenous host cell receptor (Hartland et al., 1999;
Reece et al., 2001). An alternative interpretation is that
their phenotypes may be due to (relatively subtle) Tir
binding defects of IntT914A and C937S that may be
apparent only when these mutant proteins are expressed
at natural levels on the bacterial surface. The recent iden-
tification of nucleolin as a putative endogenous host cell
receptor for EHEC intimin may provide an opportunity
to experimentally test these possibilities (Sinclair and
O'Brien, 2002).
A second goal of this study was to assess the relation-
ship between the Tir binding activity of intimin and its
ability to trigger the final step of AE lesion formation. The
eae mutations derived from the two-hybrid screen, when
expressed as full-length intimin protein on the surface of
E. coli K12, varied widely in their ability to bind to recom-
binant Tir. We found that relatively subtle defects in the
ability to bind recombinant Tir (e.g. ªtwo-thirds of wild-type
levels) were associated with significant defects in AE
lesion formation, as measured by the number and inten-
sity of actin pedestals visualized by phalloidin staining of
F-actin. Additionally, binding of intimin to recombinant Tir
correlated with the ability to trigger actin condensation.
Mutant Y882C was the sole exception, being moderately
defective for binding recombinant Tir but showing no
defect in actin signalling. There are several possible expla-
nations for this last result, including the fact that we used
recombinant EHEC Tir to assay binding but, by necessity,
used EPEC (not EHEC) for preinfection of mammalian
cells to assay actin pedestal formation. Although the
intimin 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 possible
that they differ significantly in their ability to recognize the
intimin Y882C mutant. This last exception notwithstand-
ing, we believe these results support the notion that Tir
binding is the primary activity necessary to trigger AE
lesions after E. coli type III secretion. It is possible that
intimin's sole function in actin signalling is to cluster Tir in
the plasma membrane of the eukaryotic cell. Further
investigation is required to clearly define the activities of
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Point mutants of EHEC intimin that diminish Tir binding1567
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573
intimin that are critical for colonization by attaching and
effacing pathogens.
Experimental procedures
Bacterial and tissue culture
All E. coli strains were grown in L broth at 37∞C, supplemented
with 100 mg ml-1 of ampicillin when appropriate. HEp-2 cells
were cultured in RPMI 1640 (Gibco-BRL) supplemented
with 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 generated
by Operon Technologies, or Life Technologies (see list in
Tables 3 and 4). DNA segments encoding Tir derivatives
were amplified by Pfu Turbo PCR system (Stratagene) and
inserted 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 pBTM116
to generate LexA(DB)–Tir fusions (Table 1).
Yeast transformation
Yeast strain L40 (MATa his3D200 trpI-901 leu2–3112 ade2
LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lacZ GAL4 gal80), in
which the lacZ and HIS3 promoters are fused to multimer-
ized LexA binding sites (Hollenberg et al., 1995), was used
as the reporter strain in the yeast two-hybrid system. This
strain can detect weak LexA activators as histidine prototro-
phs without the use of 3-aminotriazole. L40 was co-
transformed with pGAD424- and pBTM116-derived plasmids
by using the standard LiAc transformation procedure. Briefly,
a single colony of L40 was inoculated into 3 ml of yeast
extract peptone dextrose (YEPD) medium and grown at 30∞C
overnight with shaking. The overnight culture was then
diluted 1:50 into 50 ml of YEPD medium, bringing the OD600
to approximately 0.2, then grown at 30∞C with shaking until
OD600 reached 0.4–0.6. The cells were harvested by centrif-
ugation at 1225 g for 5 min. The cells were washed once in
sterile 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 testes
carrier DNA (Promega) were added to 100 ml of the above
yeast competent cells. Next, 600 ml of sterile 40% PEG/0.1 M
LiAc/TE (40% PEG4000 in 0.1 M LiAc/TE) was added to the
mixture and mixed by vortexing. The cells were incubated at
30∞C for 30 min with shaking. Then, 70 ml DMSO (Sigma
Chemical Co.) was added to the cells, and the cells were
heat-shocked at 42∞C for 15 min followed by incubation
on ice for 1–2 min. The cells were harvested by centrifuga-
tion at 14 000 r.p.m. in a microcentrifuge, resuspended in
500 ml of sterile distilled water, and plated onto -Trp -Leu
synthetic dropout (SD) plates to select for the desired
Quantitation of b-galactosidase activity by
ONPG liquid assays
Individual colonies of yeast transformants were inoculated
into 4 ml of SD-Trp-Leu medium and grown at 30∞C with
shaking until mid-log phase. Cells were harvested, washed
in sterile distilled water and resuspended in 2 ml of Z-buffer
(0.1 M NaPO4, pH 7.00, 10 mM KCl, 1 mM MgSO4) plus
0.27% 2-mercaptoethanol. The optical density of the cell
suspension at 600 nm was determined (A600). Then, 200 ml
of the cell suspension was added to a microcentrifuge tube
containing 300 ml of Z-buffer, 50 ml of chloroform and 50 ml of
0.05% SDS. The cells were then permeablized by vortex
mixing 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 ml
of 1 M Na2CO3. The optical density of the o-nitrophenol
product 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 eae
in the LexA(DB)–intimin fusion plasmid pHL73 (Table 2), this
region was amplified under the following mutagenic condi-
tions: 20 ng of supercoiled plasmid template, 1¥ AmpliTaq
PCR 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 mM
of each primer, 0.2 mM of either dATP or dGTP and 1 mM of
the other three deoxynucleotides, 5 units of AmpliTaq DNA
polymerase (Perkin-Elmer) in a 100 ml PCR reaction mixture
(He et al., 1996). Each reaction was preheated to 94∞C for
2 min for an initial denaturation step, followed by 35 cycles
(94∞C-1 min, 45∞C-1 min, 72∞C-1min) of amplification. The
reaction mixture was then incubated at 72∞C for five more
minutes to allow the completion of the partial products.
The primers used for mutagenic PCR were: (i) 5¢-LexA-DB
(5¢-AGGTCGTTGTCGCACGTATTGATGAC-3¢), located 152
bases upstream of EcoRI site in the coding region of the lexA
DNA binding domain; and (ii) 3¢-AdhT (5¢-GGTAGAGGTGTG-
GTCAATAAGAGCGA-3¢), located 174 bases downstream of
BamHI site in the ADH terminator sequence. A total of 500 ng
of the resulting 1131 bp PCR product was co-transformed
with 50 ng of gel-purified, gapped pHL73 (generated by
digestion of pHL73 with EcoRI and BamHI to delete the eae
coding region) into L40 cells that had been pretransformed
with pHL75 (expressing Gal4(AD)-TirM128 fusion protein;
Table 2). In vivo repair of the gapped plasmid with the
mutagenized PCR product (Muhlrad et al., 1992) resulted in
a library of pHL73 derivatives, selected on SD-Trp-Leu plates,
containing mutated LexA(DB)–Int181 sequences.
Page 12
1568 H. Liu et al.
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573
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 or
light blue colonies, suggesting a loss of interaction between
LexA(DB)–Int181 and Gal4(AD)–TirM128. Cell extracts of
such colonies were first subjected to immunoblot analysis by
using the anti-LexA mAb (Clontech) to eliminate transfor-
mants that failed to produce the full-length LexA(DB)–Int181
fusion protein. Briefly, a single yeast colony was inoculated
into 2 ml of SD-Trp-Leu broth and grown overnight at 30∞C
with shaking. The cells were harvested by centrifugation at
2500 g for 5 min. The cell pellet was then resuspended in
100 ml 1¥ SDS loading buffer (17.5 mM Tris-HCl, pH 6.8,
1.75% SDS, 1% 2-mercaptoethanol, 0.01% Bromophenol
blue) 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.
pUC19 derivative carrying CamR
Vector, CamR
Kislauskis and Dobner (1990)
Plasmids carrying full-length eae
eae in pUC19, AmpR
Identical to pHL6, but with BamHI site 3¢ to eae
eae inserted into pBC(KS) at HindIII–BamHI sites
Liu et al. (1999b)
This study
This study
Yeast two-hybrid plasmids
LexA fusion vector, TRP1, AmpR
Gal4 activation domain vector, LEU2, AmpR
pBTM116 nmd2 – produces LexA-NMD2
pBTM116 eae (1618–2802) – produces LexA–Int395
pBTM116 eae (1618–2802) – with a mutation at amino acid 932 from Cys to Ser –
produces LexA-Int395C932S
pBTM116 eae (2260–2802) – produces LexA–Int181
pBTM116 eae (2503–2802) – produces LexA–Int100
pGAD424 upf3 – produces GAD–UPF3
pGAD424 tir (1–1674) – produces GAD–TirFL
pGAD424 tir (736–1674) – produces GAD–TirM + C
pGAD424 tir (736–1119) – produces GAD–TirM128
pGAD424 tir (1–711) – produces GAD–TirN
pGAD424 tir (1096–1674) – produces GAD–TirC
Bartel and Fields
GenBank #U07647
He et al. (1997)
This study
This study
This study
This study
He et al. (1997)
This study
This study
This study
This study
This study
GST–Tir fusion plasmids
Produces GST
pGex-2T tir (736–1119) – produces GST-TirM128
pGex-2T tir (736–1708) – produces GST-TirM313
pGex-2T tir (1–711) – produces GST-TirN
pGex-2T tir (1096–1674) – produces GST-TirC
This study
Liu et al. (1999)
This study
This study
MBP–intimin fusion plasmids
Produces MBP-b-gal a-fragment
pMalc2 eae (1618–2802) – produces MBP-Int395
pMalc2 eae (1618–2802) – produces MBP-Int395C-S, with a Cys to Ser substitution
at amino acid 932
pMalc2 eae (1972–2802) – produces MBP-Int277
pMalc2 eae (2260–2802) – produces MBP-Int181
pMalc2 eae (2260–2802) – produces MBP-Int181, with a Val to Ala substitution at
amino acid 906
pMalc2 eae (2503–2802) – produces MBP-Int100
New Eng. Biolabs
Liu et al. (1999)
This study
Liu et al. (1999)
Liu et al. (1999)
Liu et al. (1999)
pMal-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 886
pMal-IntS890ApMalc2 eae (1972–2802) – produces MBP-Int277, with a Ser to Ala substitution at
amino acid 890
pMal-IntT892A pMalc2 eae (1972–2802) – produces MBP-Int277, with a Thr to Ala substitution at
amino acid 892
pMal-IntQ897ApMalc2 eae (1972–2802) – produces MBP-Int277, with a Gln to Ala substitution at
amino acid 897
pMal-IntT909ApMalc2 eae (1972–2802) – produces MBP-Int277, with a Thr to Ala substitution at
amino acid 909
pMal-IntN916ApMalc2 eae (1972–2802) – produces MBP-Int277, with a Asn to Ala substitution at
amino acid 916
pMal-IntN927A pMalc2 eae (1972–2802) – produces MBP-Int277, with a Asn to Ala substitution at
amino acid 927
This study
This study
This study
This study
This study
This study
This study
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Point mutants of EHEC intimin that diminish Tir binding1569
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573
purified from 4 ml cultures grown at 30∞C overnight in SD-
Trp-Leu broth with shaking. After centrifugation at 1225 g for
5 min, the cell pellet was washed once in 500 ml of sterile
distilled 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 vortexed
vigorously at high speed for 5 min. The supernatant was
collected after centrifugation at 16 000 g for 10 min, and the
pellet was extracted again with 200 ml of Buffer A as
described above. DNA from the pooled supernatants was
ethanol 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 carrying
pHL73 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 liquid
assays to quantitate the GAD(AD)-Tir-binding defect of the
mutants. The selected pHL73 derivatives were sequenced to
characterize the mutation(s).
Generating point mutations in full-length intimin
Full-length eae derivatives carrying the mutations identified
by the above two-hybrid analysis were cloned into high copy
number plasmids. To facilitate the transfer of eae mutations
identified in the yeast two-hybrid plasmid pHL73 to full-length
LexA–intimin fusions
5¢-cgg aat tca aca atg tac agc tta cta t-3¢
5¢-cgg aat tcg atg aac tga aaa ttg aca a-3¢
5¢-cgg aat tca cta taa aag cac cgt cgt a-3¢
5¢-cgg gat ccc ata gtt gtt gct tga tgt g-3¢
5¢-cgg gat cct tat tct aca gaa acc gca t-3¢
Gal4 AD–Tir fusions
F-tirM + C
5¢-gga aga tct caa tgc cta ttg gta atc ttg g-3¢
5¢-cgg aat tcg cgg cga cgg gta ttg tac a-3¢
5¢-cgg aat tcg cgg cga cgg gta ttg tac a-3¢
5¢-gga aga tct caa tgc cta ttg gta atc ttg g-3¢
5¢-cgg aat tca gtg gcg cat tga ttc ttg g-3¢
5¢-aac tgc agc tcc ctc taa ata aaa tga t-3¢
5¢-aac tgc agt taa cca aga atc aat gcg c-3¢
5¢-aac tgc agt taa gtc ccc aac gcc aac c-3¢
-2 to 20
-2 to 20
GST–Tir fusions
5¢-gaa gat ctg cgg cga cgg gta ttg tac a-3¢
5¢-gga aga tct atg cct att ggt aat ctt gg-3¢
5¢-gga aga tct agt ggc gca ttg att ctt gg-3¢
5¢-ccg caa ttg tta acc aag aat caa tgc gc-3¢
5¢-ccg caa ttg tta agt ccc caa cgc caa cc-3¢
5¢-ccg caa ttg ctc cct cta aat aaa atg at-3¢
5¢-gaa gat ctg cgg cga cgg gta ttg tac a-3¢
5¢-gga aga tct gct gca agt gca act gaa act g-3¢
5¢-gga aga tct ggg gta ttg aaa gat gat gt-3¢
5¢-ccg caa ttg tta caa tac ccc tga cgg aat c-3¢
5¢-ccg caa ttg ttc aat ggc ttg ctg ttt ggc-3¢
5¢-ccg caa ttg tta acc aag aat caa tgc gc-3¢
MBP–intimin fusions
5¢-cg gga tcc aac aat gta cag ctt act at-3¢
5¢-caa aag ctt tta ttc tac aga aac cgc at-3¢
eae and eae mutants
F-eaeC181 (PshAI)d
5¢-caa aag ctt cat tct aac tca ttg tgg tg-3¢
5¢-gcg act gag gtc act ttt ttt gat gaa ctg aaa att
gac aa-3¢
5¢-tta cga cac tgc ctt tac cat tca a-3¢
5¢-tat acc cgg c tta ttc tac aga aac cgc at-3¢
-30 to -11
a. F, forward (top-strand) primer; R, reverse (bottom-strand) primer. 'N' or 'C' in primer name
indicates 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 name
indicates 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 nucleotide
of the coding sequence of eae or tir.
d. This primer starts 21 nucleotides upstream of the 5¢ end of the Int181 coding region. Within
these 21 nucleotides, there is a PshAI site (underlined sequence) which facilitates the transfer
of point mutations from pHL73 into full-length eae (see Experimental procedures).
Table 3. DNA sequences of primers used in
this study.
Page 14
1570H. Liu et al.
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573
eae, three pUC19-derived plasmids carrying wild-type eae,
which differ only in flanking restriction sites or the antibiotic
marker were generated: (i) pHL109, which carries the eae
gene amplified from genomic DNA of EHEC O157:H7 strain
EDL933 by the Pfu Turbo PCR system (Stratagene) using
primers F-eae, and R-eae (Table 3), inserted into the HindIII
and BamHI sites of pUC19; (ii) the previously described
pHL6 (Liu et al., 1999), which is identical to pHL109 except
that it lacks the downstream BamHI site; and (iii) pHL112,
generated by subcloning the eae-containing HindIII–BamHI
fragment from pHL109 into pBC(KS) (Stratagene), a
chloramphenicol-resistant derivative of pUC19.
The eae mutations in pHL73 derivatives identified by the
two-hybrid screen were transferred to pHL109, pHL6 or
pHL112 by two different methods, depending on the location
of the mutations. G781D and W796R, two mutations located
5¢ to the NsiI site at position 2420–2426, were amplified from
the pHL73 derivatives by Pfu (turbo) PCR system using F-
eaeC181(PshAI) and R-eae2440 (Table 3), resulting in an
eae gene fragment carrying the mutation and flanked by
PshAI and NsiI restriction sites. The equivalent PshAI–NsiI
fragment from pHL6 was replaced with this amplified product,
resulting in pUC19-derived plasmids encoding full-length
intimin carrying either G781D or W796R. All mutations were
confirmed by DNA sequencing.
eae mutations identified in the two-hybrid screen that were
located 3¢ of the NsiI site were moved to full length eae by
replacing the NsiI–BamHI restriction fragment of pHL109,
pHL112 (or both) with the equivalent fragment from the
pHL73 derivatives. The mutations V845A, P863L, Y882C,
Q902L, G905E, V906E, N927D, V928I, V931D were moved
into pHL109, and the mutations S826P, P839L, W894R,
N911I were moved into pHL112. In all cases, the mutation
was confirmed by DNA sequencing. (Three mutations,
P839L, Y882C, and N911I, were moved into both pHL109
and pHL112, and the same mutation in either plasmid gave
roughly equivalent results for GST-Tir binding and induction
of AE lesions; data not shown. In Table 1, data shown are for
Y882C expressed from pHL109, and for P839L and N911I
expressed from pHL112.)
Site-directed mutagenesis
To generate a Cys to Ser mutation at intimin residue 932, the
coding region of the intimin C-terminal 181 amino acids was
amplified from EDL933 genomic DNA by the high-fidelity
PCR system (Boehringer Mannheim) using the primer pairs
F-eaeC181 and R-eaeC932S, which was designed to give
the Cys to Ser mutation at amino acid 932 (Table 3). The PCR
product was cleaved with NsiI and XmaI, and the resulting
250 bp fragment was used to replace the corresponding
NsiI–XmaI fragment of plasmid pHL6, a pUC19-derived plas-
mid carrying wild-type eae (Table 2). The identical mutation
was generated in pMal-Int395, which encodes an MBP–
intimin fusion protein carrying the C-terminal 396 residues
of 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-performance
liquid chromatography (HPLC)-purified and are listed in
Table 4. All mutations were confirmed by DNA sequencing.
Expression of intimin mutants on the bacterial surface
The high-copy number plasmids carrying the above eae
mutations were transformed into an E. coli K-12 strain
MC1061. The level of expression of the intimin mutants on
the bacterial surface was determined by enzyme-linked
immunoabsorbance assay (ELISA), as described previously
(Liu et al., 1999). Briefly, 1.8 ¥ 107 washed bacteria in PBS
immobilized in poly L-lysine-treated 96-well microtitre wells
(Product #3072; Becton-Dickenson Labware) and then fixed
in 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 intimin
serum (raised against the C-terminal 300 amino acids of
intimin from EHEC strain 86–24; gift of Alison O'Brien) diluted
1:500, and then horseradish peroxidase-conjugated anti-
sheep IgG (Sigma). The colorimetric peroxidase substrate
Intimin mutants
5¢-gcc att atg ctt cta tga act caa taa ctg ctt gg-3¢
5¢-gcc att ata gtt cta tga acg caa taa ctg ctt gg-3¢
5¢-gaa ctc aat agc tgc ttg gat taa aca gac atc tag-3¢
5¢-gga tta aac aga cag cta gtg agc agc gtt ctg g-3¢
5¢-gga tta aac aga cat ctg ctg agc agc gtt ctg g-3¢
5¢-cag aca tct agt gcg cag cgt tct gga gta tc-3¢
5¢-gga gta tca agc act tat gcc cta ata aca caa aac c-3¢
5¢-ctt ata acc taa tag cac aaa acc ctc ttc ctg ggg-3¢
5¢-ggt taa tgt taa tac tcc agc tgt cta tgc gg-3¢
5¢-gct tgg att aaa gcg aca tct agt gag cag-3¢
5¢-gcg ttc tgg agt atc aag cgc tta taa cct aat aac-3¢
5¢-act tat aac cta ata aca caa gcc cct ctt cct g-3¢
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 of
the complementary sequence. Underlined bases were mutated from wild type to encode
alanine residue.
c. Numbers correspond to the position of the primer in the eae coding sequence, with the ATG
start codon as positions +1 to +3.
Table 4. DNA sequences of primers used for
alanine substitutions.
Page 15
Point mutants of EHEC intimin that diminish Tir binding1571
© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 1557–1573
(TMB Microwell) was added to quantitate bound anti-intimin
antibody (Sigma 104, Sigma Chemical Co). To normalize for
variations in the number of bacteria, the same wells were
subjected to ELISA using rabbit anti-OmpA antiserum (gift of
C. Kumamoto) diluted 1:5000 and HRP-conjugated goat anti-
rabbit IgG. The sheep anti-intimin ELISA signal was divided
by the anti-OmpA ELISA signal to give an intimin:OmpA
expression ratio, and the expression ratio for each intimin
derivatives was corrected for non-specific binding by sub-
tracting the background ELISA signal of sheep anti-intimin
binding to MC1061 harbouring the negative control plasmid,
pUCAT (Kislauskis and Dobner, 1990). The resulting expres-
sion index of MC1061 harbouring wild-type intimin was
assigned a relative value of 1.00, and the expression of each
of the intimin mutants was expressed relative to this value.
We found significant experiment-to-experiment variation in
the surface expression of intimin, and therefore tested
mutants as many as seven times. Values shown in Table 1
are from a representative experiment, and are neither the
highest nor the lowest values obtained for that mutant upon
repeated determination.
Tir-binding by bacteria expressing intimin mutants
In experiments performed in parallel to those described
above, microtitre wells containing bacteria were probed with
2 mg ml-1 of GST–Tir313 or, as a control, GST. Bound GST-
Tir (or GST) was quantitated by ELISA using goat anti-GST
antibody (Pharmacia Biotech) diluted 1:1000 followed by
alkaline phosphatase-conjugated anti-goat IgG (Sigma
Chemical Co.) diluted 1:10 000 and colorimetric alkaline
phosphatase substrate, as described previously (Liu et al.,
1999). To normalize for variations in cell number, identically
treated wells were subjected to ELISA in parallel using rabbit
anti-OmpA antiserum (a gift of Carol Kumamoto) diluted
1:5000 and HRP-conjugated goat anti-rabbit antibody. Four
steps of correction and normalization were performed to
obtain a Tir-specific binding signal. First, the anti-GST-Tir
ELISA signal for each bacterial strain was divided by the anti-
OmpA ELISA signal to give a GST-Tir:OmpA ratio (or, when
GST 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 ratio
was further corrected for non-specific binding by subtracting
from it the (background) GST-Tir:OmpA ratio obtained for
MC1061 harbouring the pUCAT vector alone (Kislauskis and
Dobner, 1990). Finally, the Tir-specific binding value of each
mutant was expressed relative to that of MC1061 harbouring
pHL6, which encodes wild-type intimin and was assigned a
relative 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 mutants
to trigger actin condensation in HEp-2 cells preinfected with
an EPEC eae mutant was assessed as described previously
(Liu et al., 1999). Briefly, monolayers of HEp-2 cells were
preinfected with the EPEC eae mutant JPN15.96/pMAR7 for
3 h at 37∞C, then treated with gentamicin. Overnight cultures
of MC1061 expressing intimin were added to preinfected
monolayers and incubated for 3 h at 37∞C. After washing,
fixing, and permeabilizing the monolayers, the cells were
incubated with 5 mg ml-1 of TRITC-phalloidin and examined
by phase contrast and fluorescent microscopy (Knutton et al.,
To test for the ability of E. coli K12 expressing intimin or
intimin mutants to bind to Tir on the surface of mammalian
cells, HEp-2 cells were mock infected or preinfected with
JPN15.96/pMAR7 at a multiplicity of infection (moi) of 200,
then gentamicin treated as described above. Monolayers
were then infected at an moi of approximately 10 with E. coli
MC1061 expressing intimin or intimin mutants and, after
90 min at 37∞C, were washed six times with PBS to remove
unbound bacteria. Stably bound bacteria were quantitated by
plating for viable counts on Luria–Bertani (LB) agar with
appropriate 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 purified
as described previously (Liu et al., 1999). The concentration
of purified proteins were estimated using the BCA protein
determination kit (Pierce Chemical) and by visual comparison
of band intensity after Coomassie blue staining of SDS poly-
acrylamide gels containing dilutions of purified proteins and
known amounts of marker proteins. (The latter method was
required when trying to estimate the concentration of full-
length fusion protein when the sample contained degradation
MBP-intimin derivatives were separated by SDS-PAGE,
transferred onto a nitrocellulose membrane and blocked
with PBS + 5% milk for 1 h at room temperature (RT). The
membrane was then probed with 2 mg ml-1 of GST-TirM in
PBS + 0.2% BSA for 3 h at RT or at 4∞C overnight, and
blocked again in PBS + 5% milk for 1 h. The GST–TirM
bound to the membrane was detected by goat anti-GST
antiserum (1:1000, Pharmacia Biotech) and rabbit anti-goat
IgG conjugated to alkaline phosphatase (Sigma Chemical
Modified ELISA to detect the interactions between
MBP–intimin and GST–Tir fusions
To obtain quantitative measures of the binding activity of
MBP–intimin fusion proteins to GST–Tir derivatives, 96-well
Linbro microtitre plates (ICN/Flow Biomedicals) were coated
with 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∞C
and probed with 2 mg ml-1 of GST-TirM in PBS + 0.2% BSA
for 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 goat
anti-GST antiserum (1:1000, Pharmacia Biotech) and rabbit
anti-goat IgG conjugated to alkaline phosphatase (Sigma
Chemical Co.).
Interpreting the EHEC sequence based on the EPEC
intimin–Tir complex crystal structure
The complex of EHEC intimin and Tir was modelled based
on the crystal structure of the EPEC intimin and Tir (Luo


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"Spectre" James Bond Film Unveils Trailer
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"The Walking Dead" Spin-Off TV Series Unveils Title
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"Transformers" Getting Lots More Sequels and Spin-Offs
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First Look at Ryan Reynolds in "Deadpool"
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Trailer for Jake Gyllenhaal's "Southpaw" Boxing Film
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Trailer for "Absolution" Thriller, with Steven Seagal and Vinnie Jones
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Julia Louis-Dreyfus in Talks for American Remake of "Force Majeure"
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Trailer for Kit Harington's "Spooks: The Greater Good"
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First Look at Jesse Eisenberg as Lex Luthor in "Batman v Superman: Dawn of Justice"
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Trailer for Arnold Schwarzenegger's "Maggie" Zombie Film
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First Clear Look at The Thing From "Fantastic Four" Reboot
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Idris Elba May Play Villain in "Star Trek 3"
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New Photos of Ben Stiller and Owen Wilson From "Zoolander 2"
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New Trailer for "Entourage" Movie
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Poster for "Fantastic Four" Reboot
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Trailer for Dustin Hoffman's "Boychoir" Film
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Jennifer Lawrence Says "X-Men: Apocalypse" is Her Last "X-Men" Film
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"Avengers: Infinity Wars" Officially Replaces Director Joss Whedon
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Vivica A. Fox Returns for "Independence Day 2"
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Poster for "Entourage" Movie
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Tom Cruise's "Mission: Impossible 5" Trailer is Here!
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Neill Blomkamp Says "Alien 5" Won't Interfere With "Prometheus" Franchise
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Quentin Tarantino Says "Prometheus" Had Lots of Dumb Stuff
Posted: January 5th, 2013 by
WorstPreviews.com Staff
While promoting "Django Unchained," director Quentin Tarantino spoke to Craig Ferguson, revealing what he thought about Ridley Scott's "Prometheus."
I saw 'Prometheus.' I loved it and I was disappointed at the same time,
" explained Tarantino. "
On one hand I was a little disappointed about it. On the other hand, it was actually kind of cool to see such a big deal, serious, science fiction epic by a director like Ridley Scott.
He continued: "
There were parts of it I actually did like and over-all the experience was really cool having been in it. There was also a lot of dumb stuff in it, though.
Tarantino didn't name all parts that he felt were dumb, but mentioned one scene in which the biologist approaches the "space cobra." The director couldn't believe that a character would behave the way he did around an alien creature. Check out the video below to hear the full response (starts at 4:48 mark).
You must be registered to post comments.
Turn On
on January 5th, 2013 at 7:21:31 AM
just saw django, that sh*t was righteous!
on January 5th, 2013 at 7:24:25 AM
I always felt the same way about Will Smith sockin that alien in the face in ID4
Remove that from the movie and add a les scene with Goldblooms wife and Vivica Fox and you got yourself a winner
on January 5th, 2013 at 7:40:42 AM
^the will smith son was actually the most annoying thing of that movie...
on January 5th, 2013 at 8:08:20 AM
Once again Tarantino being a wise mouth.nothing special about django.same revenge bullsh*t.only reason I liked it was cause most movies sucked this year.nothing more
Dark knight rises what a let down.better than the last one though.
Will check out looper today hopefully doesn't suck.
Abraham Lincoln vampire hunter worth a watch
on January 5th, 2013 at 8:09:15 AM
Next week dredd on DVD
on January 5th, 2013 at 8:09:25 AM
So, out there are the Roswell files.
Mr. Blonde
on January 5th, 2013 at 8:14:25 AM
Yeah, those two idiot scientists were nothing but alien bait the moment they left the landing party. Agree with Tarantino but I would have expected him to expand on his comment a bit more. Like those dumb motherf*ckers deserved to get their motherf*cking faces melted off for being so motherf*cking retarded to even mess with a motherf*cking creature from outer space. Or something along those lines.
on January 5th, 2013 at 8:31:20 AM
"The director couldn't believe that a character would behave the way he did around an alien creature"
No one could...
on January 5th, 2013 at 8:37:48 AM
Anyway, I have realize that Blade Runner is nothing more than a piece of sh*t painted with gold.
A f*cking mess.
But I've seen it many times, though, and have the Final Cut on DVD also.
It's a mess of a movie. IMO.
on January 5th, 2013 at 8:41:58 AM
Dredd is good. But could have been better even with that budget.
on January 5th, 2013 at 9:47:12 AM
There was not one intelligent point made in Prometheus, not one. It did absolutely nothing new for the genre or the Alien series. The characters (all except David) were extremely stupid, the editing was terrible (with many explanations for plot holes big and small left on the editing room floor), and the story (which had a great premise and huge potential) was dumb in the long run.
What's there to like QT and others?
on January 5th, 2013 at 10:02:33 AM
Id have to watch it again, 6 months of therapy was the only thing to wipe it from my mind.
on January 5th, 2013 at 10:36:44 AM
Just a thought:
I wish Ridley would have replaced Charlize Theron's character with Guy Pearce. He could have portrayed Weyland's son and old man Weyland giving the film a pretty cool acting dynamic while also providing a reason for the terrible make up on Guy. Maybe there could have been an underlying mystery about Pearce possibly being a clone which Weyland might have produced in his desperate quest to cheat death. Pearce could loathe his father because he knows how much alike they are, but he doesn't know the real reason they share so many similarities until he goes on the mission to find the Engineers. It would add another level to this Creator theme of the film and could be a great source for the main conflict of the film. This entire mission could be a backdrop for David getting revenge against his creator and clone brother. Maybe he always resented Pearce's character because as a clone he could experience emotions and share a connection with his father, something David's character could not. Once they arrive on the planet David's plot for revenge against his maker soon turns into a much bigger plan to wipeout mankind by completing the Engineers' work. In trade for assisting the remaining Engineer David seeks the knowledge to create life and father it in his own image.
on January 5th, 2013 at 12:00:48 PM
Guy Pearce was misused in that movie,I thought from the viral video that we'll see a younger version which was so dominant in that viral clip but instead we got this horrendous make up old version which was pointless as well as charlize theron's character.
on January 5th, 2013 at 12:29:54 PM
@ Tarantino Says
Prometheus Had Lots of Dumb Stuff
like what...
silly childish characters that curse non stop and talk just to shove cool down your throat
on January 5th, 2013 at 12:36:55 PM
I enjoyed Prometheus tbh, yes it was faulty but hey ...people saying we wanted to see this and that and stuff , but it's gonna be a trilogy ...right ? First one is always gonna be a bit boring like the first lord of the rings then the sequels are better with hopefully a more interesting story line, I think it's great that a movie can create such a mixed set of results for people ...that's what's good about it . Everyone sees it different way. I can't remember a film creating such a mixed feel to it ?
on January 5th, 2013 at 12:51:00 PM
I'd agree with QT if he a)his films weren't full of even dumber sh*t, and b) he could actually specify what was dumb without simply repeating what he read on the internet. Then again, this is the guy who readily admits he rips off other people's films to make his own movies.
on January 5th, 2013 at 12:55:53 PM
the one you've all been waiting for
on January 5th, 2013 at 1:08:48 PM
the ones you've all been waiting for
on January 5th, 2013 at 3:01:35 PM
^^^^haven't been waiting for any of those. And while ill say QT has merit here, I'd still bet he is just using the cliche complaints out there in an attempt to puff up his own work in better lighting. But the really pathetic thing is none of hismovies are even a fraction as good as ridley scotts. HRS has more talent leaving his body during a morning constitutional than QT could ever dream of. Put makeup on your sh*t movies all you want QT, but there are still sh*t. And you are one majorly f*cked up human being.
on January 5th, 2013 at 3:16:26 PM
on January 5th, 2013 at 3:46:46 PM
@ vincere01
haven't been waiting for any of those ??
let me guess
you wanna watch THE MASTER
SILVER LININGS PLAYBOOK maybe..
on January 5th, 2013 at 4:13:28 PM
I'd be up for the Master lol
on January 5th, 2013 at 4:50:27 PM
just saw Life of Pi
Great movie,
oh sorry the wrong post...
just didn't saw Django
on January 5th, 2013 at 6:26:40 PM
I never liked QT...Pulp fiction is one of the most overrated movies of all time.
on January 5th, 2013 at 8:00:26 PM
Death Proof had lots of dumb stuff too, like Title card to credits.
on January 5th, 2013 at 10:54:38 PM
I love everybody saying he should expand on his comment. You know why it's stupid, you know why he's saying it.
Just agree, you know why you'd be doing it anyway.
on January 5th, 2013 at 11:23:24 PM
"I never liked QT...Pulp fiction is one of the most overrated movies of all time."
And it goes without saying, but I shall it anyway, QT is up there in the top five of most over-rated directors of all time.
If you want a poster boy for self-indulgence, then look no further. For those of you who want to make movies the Tarantino way, follow this simple recipe:
1. Drink lots of coffee.
2. Watch 20 B-movies in a row without sleeping.
3. Write a script copying sequences verbatim.
4. Cut up script and rearrange the sequences at random.
5. Insert conversation you had with racist/sexist wino on the bus into script.
6. Record for 2 hours as you channel surf through white trash oldies radio. This will be your soundtrack.
7. Send script to Harvey Weinstein.
8. Get a big head (literally and figuratively).
9. Direct with big head firmly planted up ass. Tell actors to treat your dialogue like its from the Bible.
10. Make sure the running time is over 2 hours.
11. Go to imdb.com and look up obscure films. Claim them as your influences.
on January 5th, 2013 at 11:56:09 PM
Here's some new news for ya, Alex.
QT annoyed with criticism over Django's violence
So, when do I get paid?
on January 6th, 2013 at 12:15:52 AM
on January 6th, 2013 at 12:23:57 AM
"5. Insert conversation you had with racist/sexist wino on the bus into script."
But you forgot to add "Be sure to sift through dictionary for a whole day for said wino sequences". I'm sure you could pretty that up, but don't forget it.
on January 6th, 2013 at 2:39:00 AM
if u think br is a mess you are a fool.
on January 6th, 2013 at 3:22:14 AM
I just saw django unchained, bit disappointed.
And about BR, Deaftone, of course the final cut got many editing corrections, but the other versions are nonsense.
The plot is quiet simple, very generic, what makes this flick impressive is bits of good direction and beautiful shots, great fx, outstanding mis� en scene, great music.
Again, I must repeat myself I think, story wise wasn't that brilliant, any single short story written by Phil d*ck is better in many ways.
I haven't read the book but I know the flick is quiet different, beginning by taking the name from an novel-screenplay written by none other but William S. Burroughs.
So there you have it.
P.S. If it wasn't a mess since the beginning, it wouldn't had need as many versions and editions as it has.
It's a mess. I've seen it many, many times, as to be as secure as I feel and know I am.
Of course, the point is that BR isn't quite pretentious as Prometheus is, but well, it's are to translate accurately a work of d*ck in a very faithful way.
Yes, indeed, there are sometimes that pretentiousness do touch some thing that f*ck up pretension, then comes ambition, but well, is a hell of a fight to create something as a novel or a movie, indeed.
on January 6th, 2013 at 3:27:22 AM
Django Unchained was disappointing in the sense that I ain't going to see it on the theater.
on January 6th, 2013 at 3:28:08 AM
it's preposterous to even dare to comment about what was so disappointing about it.
on January 6th, 2013 at 4:17:12 AM
on January 6th, 2013 at 5:06:06 AM
and MINK, I like #five
*Imma poet and didnt know it*
on January 6th, 2013 at 9:30:10 AM
Unga Bunga!? lol
on January 6th, 2013 at 1:34:09 PM
@pornfly: lol.
on January 7th, 2013 at 1:19:37 AM
abe lincoln vampire hunter?? for real???
dark knight rises over dark knight (both of which sucked IYO)???
a dredd dvd release drop???
nice post gay boi
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|JJ Redick joins the Bucks
|Written by Admin
|Friday, 22 February 2013 11:01
Milwaukee acquired shooting guard J.J. Redick from the Orlando Magic along with point guard Ishmael Smith and center Gustavo Ayon in exchange for backup point guard Beno Udrih and youngsters Tobias Harris and Doron Lamb.
General manager John Hammond said Friday the moves were a ``win-now'' proposition.
Milwaukee has gone 10-11 under Jim Boylan since Scott Skiles left. The Bucks take a 26-27 record into their game Saturday night against Atlanta, good enough for eighth in the conference.


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Arizona Cardinals
Kurt Warner throws passes to Larry Fitzgerald
Here's hoping Carson Palmer makes us stop tearing up at sights like this.
But until then, seeing Kurt Warner -- gloves and all -- tossing a football to Larry Fitzgerald at University of Phoenix Stadium is likely enough to make any Arizona Cardinals fan a bit emotional.
After all, the two combined for hundreds of completions and 39 touchdowns, and the receiver has not experienced nearly the same level of success since his former quarterback hung up his cleats.
You can thank Cardinals VP of Media Relations Mark Dalton for the video...as well as any nostalgia you may be feeling.
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- Charles Robinson, Yahoo! Sports NFL reporter - Tuesday March 24Charles Robinson believes that Adrian Peterson's relationship with the Vikings will only get worse.
- Sal Paolantonio- ESPN NFL analyst - Tuesday March 24Bruce Arians told Sal Paolantonio that there are "no serious discussions" about trading for Adrian
- Herm Edwards, ESPN NFL insider - Tuesday March 24What are the chances Adrian Peterson plays for the AZ Cardinals this season?


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Break, Break, Break
Break, Break, Break Themes
Break, Break, Break Themes
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The repetition in "Break, Break, Break" both suggests the consistency of the speaker's grief and also its slow-but-steady evolution. The second line of the final stanza sounds like the second...
Language and Communication
The speaker of "Break, Break, Break" tells us that he can't express his thoughts or his grief, but then he goes on for three more lyrical, lovely stanzas about how much he misses his dead friend....
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