Báo cáo khoa học: Cytoskeleton-modulating effectors of enteropathogenic and enterohemorrhagicEscherichia coli: a case for EspB as an intrinsically less-ordered effector pptx

MINIREVIEW
Cytoskeleton-modulating effectors of enteropathogenic
and enterohemorrhagic Escherichia coli: a case for EspB as
an intrinsically less-ordered effector
Daizo Hamada
1
, Mitsuhide Hamaguchi
2
, Kayo N. Suzuki
3
, Ikuhiro Sakata
2
and Itaru Yanagihara
4
1 Division of Structural Biology (G-COE), Graduate School of Medicine, Kobe University, Japan
2 Department of Emergency Critical Care Medicine, Kinki University, Osaka, Japan
3 Laboratory of Cell Migration, RIKEN, Center for Developmental Biology, Kobe, Japan
4 Department of Developmental Medicine, Osaka Medical Center for Maternal and Child Health, Izumi, Japan
Introduction
Gram-negative pathogenic bacteria maintain a type III
secretion system (T3SS) that functions in secreting vir-
ulence factors directly into the cytosolic space of host
cells [1]. Among such virulence factors, several effector
proteins influence the morphology of actin filaments
that maintain host-cell morphology and cell–cell con-
tacts.
In the case of enteropathogenic or enterohemor-
rhagic Escherichia coli (EPEC or EHEC, respectively),
effectors involved in actin reorganization include
E. coli secreted protein (Esp)B [2,3], EspF
U

[4–6] and
EspL2 [7,8]. By interacting with host proteins involved
in the regulation of actin morphology, these factors
control morphological changes in filaments, thereby
allowing the formation of actin-based pedestals that
underlie bacterial attachment sites on the host-cell
membrane.
The work of our group focuses on the role of EspB
in host-cell actin reorganization [9], in particular, how
the conformational properties of EspB contribute
Keywords
actin reorganization; adherence junction;
alpha-catenin; bacterial infection; disorder
prediction; intrinsically disordered; molten
globule; multifunctional protein; pedestal
formation; type III secretion system
Correspondence
D. Hamada, Division of Structural Biology
(G-COE), Department of Biochemistry and
Molecular Biology, Graduate School of
Medicine, Kobe University, 7-5-1
Kusunoki-cho, Chuo-ku, Kobe 650-0017,
Japan
Fax: +81 78 382 5816
Tel: +81 78 382 5817
E-mail:
(Received 14 December 2009, revised 13
January 2010, accepted 4 February 2010)
doi:10.1111/j.1742-4658.2010.07655.x
Enterohemorrhagic and enteropathogenic Escherichia coli produce various

effector proteins that are directly injected into the host-cell cytosol through
the type III secretion system. E. coli secreted protein (Esp)B is one such
effector protein, and affects host-cell morphology by reorganizing actin net-
works. Unlike most globular proteins that have well-ordered, rigid struc-
tures, the structures of type III secretion system effectors from pathogenic
Gram-negative bacteria, including EspB, are often less well-ordered. This
minireview focuses on the functional relationship between the structural
properties of these proteins and their roles in type III secretion system-
associated pathogenesis.
Abbreviations
EHEC, enterohemorrhagic Escherichia coli; EPEC, enteropathogenic Escherichia coli; Esp, E. coli secreted protein; T3SS, type III secretion
system; Tir, translocated intimin receptor.
FEBS Journal 277 (2010) 2409–2415 ª 2010 The Authors Journal compilation ª 2010 FEBS 2409
to the multifunctionality of this protein [10]. EspB
binds to various host proteins including a-catenin [2],
a
1
-antitrypsin [11] and myosin [12]. To create a pore
on host-cell membranes, EspB and EspD bacterial
proteins form a complex that binds to an EspA needle
on the bacterial membrane [13]. Assembly of this
complex results in a conduit that links bacterial and
host-cell membranes.
In this minireview, we describe the multifunctional
roles that EspB plays in pedestal formation and in medi-
ating morphological changes in infected host cells. We
compare the structural properties of EspB and other
T3SS effectors in various pathogenic Gram-negative
bacteria and describe how the ‘intrinsically less-ordered’
nature of these effectors contributes to pathogenesis.

EspB as an effector of actin filament
reorganization
The EspB (or EarB) gene product was first identified as
an important factor in EPEC attachment [14] and later
characterized as a T3SS-secreted protein required for
signal transduction [15]. During its function in attach-
ment, EspB associates with EspD [13] at the tip of a
hollow EspA filament formed on the bacterial cell sur-
face [13], resulting in the formation of a pore on a host
cell (Fig. 1) that serves as a conduit between bacterial
and host-cell membranes. Pore formation allows the
secretion of T3SS virulence factors into host cell.
In addition, EspB functions as a signal transducer
or effector. It is secreted via a T3SS needle into the
host-cell cytosol, where it participates in the rearrange-
ment of actin molecules that promote morphological
changes in host cells and pedestal formation [16,17].
Although EspB has the potential to form pore struc-
tures together with EspD, EspA is required to translo-
cate this protein into the host cytosol [18]. As an
effector, EspB binds to host proteins, including a-cate-
nin [2] and myosin [12], that regulate cytoskeletal mor-
phology by controlling actin network formation. After
binding, EspB redirects the activity of these regulator
proteins to generate actin-based cytoskeletal pedestals
that are the basis for EHEC and EPEC attachment
sites (Fig. 1). EspB is therefore required both as a
pore-forming protein and a signal effector during
EHEC and EPEC pathogenesis.
When bound together, EHEC EspB promotes the

action of a-catenin in bundling actin filaments, in
opposition to the action of actin-related protein 2 ⁄ 3in
promoting actin filament branching [9]. This activity is
consistent with EspB ⁄ a-catenin colocalization at pedes-
tals, as well as the role of EspB in reorganizing actin
filaments and host proteins associated with cell mor-
phology. In binding to a-catenin, EspB also promotes
the dissociation of a-catenin from the E-cadherin ⁄
b-catenin ⁄ a-catenin complex at cell–cell adherence
junctions [9], which probably leads to the destabiliza-
tion of cell contacts [19] and facilitates bacterial
penetration through intestinal epithelium.
Importantly, EspB binds to the C-terminal vinculin
homology domain of a-catenin, whereas formation of
a-catenin ⁄ b-catenin ⁄ E-cadherin complexes at adher-
ence junctions requires the N-terminal vinculin homol-
ogy domain of a-catenin [9]. Based on these
interactions, it was hypothesized that conformational
changes in a-catenin mediated by EspB, rather than
EspB-blocking interactions with b-catenin, lead to the
dissociation of a-catenin from adherence junction
complexes (Fig. 1).
EspB also interacts with the actin-binding domain of
several myosin proteins, including myosin-1a, -1c, -2,
-5, -6 and -10 [12]. By inhibiting interactions between
myosins and actins, EspB can prevent both the initia-
tion of phagocytosis and the production of microvillus
effacing [12]. It has been reported that deletion of the
central domain (amino acids 159–218) of EspB creates
a mutant protein that cannot bind to myosin-1c; never-

theless, an EPEC mutant strain carrying this EspB
deletion translocated virulence factors to host cells and
EHEC
Adherence
junction
E-cadherin
-catenin
-catenin
EspB
Secretion of EspB
through T3SS
Dissociation
of -catenin
Bundling of
actin filaments
Actin filaments
Accumulation of bundled
actin filaments for
pedestal formation
EspB/D
Pore
Fig. 1. Schematic representation of roles of EspB. EspB secreted
into host-cell cytosol binds to the C-terminal region of the a-catenin,
destabilizing E-cadherin ⁄ b-catenin ⁄ a-catenin complexes at adher-
ence junctions that mediate cell–cell contacts and cytoskeletal mor-
phology. Binding of EspB to the C-terminal region of a-catenin
promotes the dissociation of N-terminal interactions of a-catenin
with b-catenin. Thu, during this process, EspB does not merely
compete with b-catenin for a-catenin binding, but in fact induces a
conformational change in the N-terminal region of a-catenin by bind-

ing to the C-terminal region. EspB-bound a-catenin shows
enhanced affinity with actin filaments and also promotes bundling
of actin filaments that accumulate at pedestals formed underneath
the attachment site of bacterial cell.
EspB as an intrinsically less-ordered effector D. Hamada et al.
2410 FEBS Journal 277 (2010) 2409–2415 ª 2010 The Authors Journal compilation ª 2010 FEBS
induced actin reorganization [12]. These results are
consistent with experiments that map the a-catenin-
binding domain of the EHEC EspB to the N-terminus
(amino acids 1–108) [2].
Structural properties of EspB and other
T3SS effectors
3D structures of numerous proteins associated with
EHEC or EPEC T3SS have been solved [20–28] using
X-ray crystallography, solution NMR [29–31] and
cryo-EM [32]. However, because of the tendency of
this protein to assume a less-ordered conformation, the
structural properties of EspB are currently unknown.
EspB consists of a substantial amount of a-helical
structure, but lacks rigid structures commonly found
in globular proteins [10], and therefore is classified as a
‘natively partially folded protein.’
Recently, various functional proteins have been
found that maintain almost completely disordered
structures even under native conditions. These proteins
are called ‘natively unfolded’ or ‘intrinsically disor-
dered’ proteins [33,34]. EspB is basically unfolded but
maintains some secondary structures. The structure is
therefore more similar to the partially folded or ‘mol-
ten globule’ states of globular proteins that accumulate

during folding kinetics [35,36]. As shown in Fig. 2A,
a far-UV CD spectrum shows that EspB contains
a-helical structures but with less-ordered tertiary folds
according to the less-dispersed signals in a
15
N–
1
H
HSQC spectrum (Fig. 2B) [10]. For this reason, EspB
protein is classified as ‘natively partially folded’, rather
than ‘natively unfolded’ or ‘intrinsically disordered’
[10]. A similar structural property has been observed
with PopD which is a homolog of EspB expressed by
Pseudomonas aeruginosa [37]. It should be noted here
that, according to the original definition by Ohgushi &
Wada [35], the ‘molten globule’ is a partially folded
intermediate state with a significant amount of second-
ary structure, similar to the tightly packed native state,
but lacks tertiary contacts. The molten globule state
has been considered to be a relatively stable intermedi-
ate state that is accumulated during kinetic or equilib-
rium refolding or unfolding of a globular protein, in
contrast to the well-ordered native structures with
rigid secondary and tertiary structures under near
native conditions or fully unfolded structure without
ordered conformations. Use of the term ‘molten glob-
ule’ therefore sounds as if it is an intermediate state
accumulated during the folding reaction into the
tightly packed ordered structures with well-ordered
secondary and tertiary structures. However, EspB

assumes a ‘partially folded’ structure under native con-
ditions and does not form a tightly packed native
structure by itself. To clarify that the structure of
EspB is the native state rather than the folding inter-
mediate, we do not use terms such as ‘natively molten
globule’ or intrinsically molten globule’, particularly
for EspB.
Various algorithms can predict disorder regions of
proteins from their amino acid sequences and the Pre-
dictor of Naturally Disordered Regions (PONDR
Ò
;
) algorithm is one of them [38–
40]. This algorithm suggests that EspB contains sub-
stantial amounts of disordered and some ordered
regions (Fig. 3). However, it should be noted that the
predicted ordered regions in this calculation do not
neccessarily mean that the regions are folded into
well-ordered rigid structures as usually observed for
globular proteins and, in this case, they assume less-
ordred partially folded structures [10]. Interestingly,
the putative a-catenin- (1–108 in EHEC EspB) [2] and
myosin-binding regions (159–218 in EPEC EspB) [12]
of EspB overlap with regions predicted to assume
ordered conformations (Fig. 3). These data suggest
that the a-helical structures found experimentally in
EspB coincide with ordered regions, and that an abil-
ity to assume an a-helical structure may be involved
in the recognition of a-catenin or myosin host target
proteins.

EspB binds to various proteins including EspA and
EspD from bacteria and a
1
-antitrypsin and a-catenin
from host cells. We also found that host proteins other
than a-catenin that are involved in the regulation
of cytoskeletal morphology can bind to EspB
(M. Hamaguchi, I. Yanagihara, K. N. Suzuki & D.
Hamada, unpublished data). This indicates that EspB
is a typical multifunctional protein. The 3D structures
15
10
5
0
–5
–10
[
q
] × 10
–3
(deg·cm
2
dmol
–1
)
250230210190
Wavelength (nm)
1
H (ppm)
15

N (ppm)
98
7
610
130
120
110
A
B
Fig. 2. Structural properties of EspB. Far-UV CD and
1
H-
15
N HSQC
spectra of EspB obtained at 20 °C, pH 7.0. EspB assumes a signifi-
cant amount of a-helical structure according to CD (A), but less-dis-
persed signals are observed in HSQC spectra (B), suggesting a lack
of rigid conformation. These data indicate that EspB assumes a
‘natively partially folded’ conformation, similar to the ‘molten glob-
ule’ state. Spectra-based figures are reproduced from Hmada et al.
[10] with permission from the publisher.
D. Hamada et al. EspB as an intrinsically less-ordered effector
FEBS Journal 277 (2010) 2409–2415 ª 2010 The Authors Journal compilation ª 2010 FEBS 2411
of these EspB target proteins differ significantly
(M. Hamaguchi, I. Yanagihara, K. N. Suzuki & D.
Hamada, unpublished data). Therefore, it is highly
quiestionable how this protein with only 330 amino
acid residues manages to recognize these different
targets. The structural flexibility of EspB caused by the
formation of partially folded structures could be

advantageous for its multifunctional properties because
its association with various targets of different mole-
cular dimensions and binding surfaces would be facili-
tated as different conformations can be assumed.
In T3SS proteins from bacteria other than EHEC
and EPEC, less-ordered proteins such as IpaC from
Shigella flexneri, SipC from Salmonella, PopD from
Pseudomonas aeruginosa or YopD from Yersinia pestis,
demonstrate functions homologous to EspB (Fig. 3).
In complex with IpaB, IpaC forms a pore on host-cell
membranes and is also the effector that triggers actin
polymerization during the formation of filopodia and
lamellipodia [41–43]. SipC is involved in nucleation
and bundling of actin filaments via direct binding to
actin [44], whereas PopD from Pseudomonas aeruginosa
or YopD from Yersinia species also form a pore
complex, in this case with PopB [45] or YopB [46],
respectively. Similar to EspB [10], some of these other
proteins have also been shown to assume disordered or
partially folded conformations under native conditions
[47,48].
T3SS effector proteins that are not homologous to
EspB have also been shown to exhibit ‘natively
unfolded’ structures. For example, Yersinia YopE is a
cytotoxin that uses GTPase-activating protein activity
to target the Rho pathway to induce disruption of
actin microfilament structures [49]. The structured
region of YopE, which has been resolved using crystal-
lography, has been shown to correspond to a GTPase
activator [50]. By contrast, other parts of this protein

are disordered entirely in solution, but can assume an
ordered structure upon binding to a chaperone [51].
Both EHEC and EPEC encode the translocated inti-
min receptor (Tir) protein, which localizes to plasma
membranes and forms clusters of proteins when bound
to the bacterial outer membrane protein, intimin
[29,30]. Tir has also been shown to bind the bacterial
EspF
U
⁄ Wiskott–Aldrich syndrome protein complex
through either the insulin receptor tyrosine kinase sub-
strate or its homolog, the 53-kDa insulin receptor sub-
strate protein that regulates cytoskeletal organization
[4,5]. According to CD spectra, Tir is largely unstruc-
tured in solution [52]; the PONDR
Ò
algorithm also
predicts that large regions of Tir and EspF
U
have a
propensity to form disordered structures (Fig. 3) [52].
This collection of findings suggests that relative
structural disorder may be a common feature of T3SS
effectors. Less-structured proteins may be favoured for
secretion through the narrow T3SS pore, as suggested
for flagella T3SS [53], and may also better serve the
multiple roles required during pathogenesis.
1.0
0.5
0.0

PONDR score
1.0
0.5
0.0
PONDR score
1.0
0.5
0.0
PONDR score
1.0
0.5
0.0
PONDR score
3002001000
Residue number
3002001000
Residue number
3002001000
Residue number
4003002001000
Residue number
EspB
YopD
EspFu
α
1-Antitrypsin
EPEC
EHEC
1.0
0.5

0.0
PONDR score
1.0
0.5
0.0
PONDR score
4003002001000
Residue number
4003002001000
Residue number
IpaC
SipC
1.0
0.8
0.6
0.4
0.2
0.0
PONDR score
1.0
0.8
0.6
0.4
0.2
0.0
PONDR score
6004002000
Residue number
Tir
200150100500

Residue number
p27kip1
Fig. 3. Disorder in various T3SS effectors. Predictions of EspB
from EHEC (solid line) and EPEC (dotted line), IpaC from Shigella,
YopD and YopE from Yersinia, SipC from Salmonella and EspF
U
and Tir from EHEC. The predictions derived from PONDR
Ò
[38–40].
Regions with a PONDR score > 0.5 are predicted to be disordered
and those with a score < 0.5 are predicted to be ordered. The
PONDR analysis for a
1
-antitrypsin (a typical natively folded protein
with a serpentine fold) and for cyclin-dependent kinase inhibitor
p27kip1 (a typical natively unfolded protein) [54] are shown for
comparison. Predictions for effector proteins and p27kip1 shown
larger regions of predicted disorder relative to natively folded
a
1
-antitrypsin. It should be noted that the predicted ordered regions
do not neccessarily assume rigid folded structures usually observed
for globular proteins and can form partially folded structures similar
to the molten globule state [10].
EspB as an intrinsically less-ordered effector D. Hamada et al.
2412 FEBS Journal 277 (2010) 2409–2415 ª 2010 The Authors Journal compilation ª 2010 FEBS
Conclusions
We have reviewed the role of EspB as an EHEC ⁄ EPEC
effector and explained how the ‘natively partially
folded structure’ of this protein contributes to its multi-

functionality. Although a lower proportion of intrinsi-
cally disordered proteins is encoded in bacterial
genomes relative to eukaryotes [54], structural disorder
has also been observed in other T3SS effectors. Like
pathogenic viruses [55], these bacterial effectors may
have evolved to mimic host protein structural proper-
ties in order to regulate the target proteins of host cells.
Structural disorder in T3SS effectors may be also an
important factor for secretion through T3SS needles.
Various EHEC or EPEC effectors, including EspB,
EspF
U
and EspL2, regulate host-cell actin networks.
In the future, clarification of the interplay between
these effectors and a detailed analysis of EspB in com-
plex with host targets will provide important insight
into these interactions. Via the EspA-mediated T3SS
apparatus, EspB is guided to form pore structures in
complex with EspD, resulting in a conduit between
bacterial and host cell membranes. Structural models
depicting this initial stage of infection by bacteria
should be allow better understanding of pathogenetic
mechanisms of EHEC and EPEC.
Acknowledgements
This work was supported by Grants-in-Aid for the
Global COE program A08 from the MEXT, Japan,
and Grant-in-Aid for Young Scientists (B) from Japan
Society for the Promotion of Science (to DH).
References
1 Galan JE & Collmer A (1999) Type III secretion

machines: bacterial devices for protein delivery into host
cells. Science 284, 1322–1328.
2 Kodama T, Akeda Y, Kono G, Takahashi A, Imura K,
Iida T & Honda T (2002) The EspB protein of entero-
haemorrhagic Escherichia coli interacts directly with
a-catenin. Cell Microbiol 4, 213–222.
3 Taylor KA, O’Connell CB, Luther PW & Donnenberg
MS (1998) The EspB protein of enteropathogenic
Escherichia coli is targeted to the cytoplasm of infected
HeLa cells. Infect Immun 66, 5501–5507.
4 Vingadassalom D, Kazlauskas A, Skehan B, Cheng
H-C, Magoun L, Robbins D, Rosen MT, Saksela K &
Leong JM (2009) Insulin receptor tyrosine kinase sub-
strate links the E. coli O157:H7 actin assembly effectors
Tir and EspF
U
during pedestal formation. Proc Natl
Acad Sci USA 106, 6754–6759.
5 Weiss SM, Ladwein M, Schmidt D, Ehinger J, Lommel
S, Sta
¨
ding K, Beutling U, Disanza A, Frank R, Ja
¨
nsch
L et al. (2009) IRSp53 links the enterohemorrhagic
E. coli effectors Tir and EspF
U
for actin pedestal for-
mation. Cell Host Microbe 5, 244–258.
6 Campellone KG (2010) Cytoskeleton-modulating

effectors of enteropathogenic and enterohaemorrhagic
Escherichia coli: Tir, EspFU and actin pedestal assem-
bly. FEBS J 277, 2390–2402.
7 Miyahara A, Nakanishi N, Ooka T, Hayashi T, Sugim-
oto N & Tobe T (2009) Enterohemorrhagic Escherichi-
a coli effector EspL2 induces actin microfilament
aggregation through annexin 2 activation. Cell Micro-
biol 11, 337–350.
8 Tobe T (2010) Cytoskeleton-modulating effectors
of enteropathogenic and enterohaemorrhagic
Escherichia coli: role of EspL2 in adherence and an
alternative pathway for modulating cytoskeleton
through Annexin A2 function. FEBS J 277, 2403–2408.
9 Hamaguchi M, Hamada D, Suzuki KN, Sakata I &
Yanagihara I (2008) Molecular basis of actin reorgani-
zation promoted by binding of enterohaemorrhagic
Escherichia coli EspB to a-catenin. FEBS J 275, 6260–
6267.
10 Hamada D, Kato T, Ikegami T, Suzuki KN, Hayashi
M, Murooka Y, Honda T & Yanagihara I (2005) EspB
from enterohaemorrhagic Escherichia coli is a natively
partially folded protein. FEBS J 272, 756–768.
11 Knappstein S, Ide T, Schmidt MA & Heusipp G (2004)
a
1
-Antitrypsin binds to and interferes with functionality
of EspB from atypical and typical enteropathogenic
Escherichia coli strains. Infect Immun 72, 4344–4350.
12 Iizumi Y, Sagara H, Kabe Y, Azuma M, Kume K,
Ogawa M, Nagai T, Gillespie PG, Sasakawa C &

Handa H (2007) The enteropathogenic E. coli effector
EspB facillitates microvillus effacing and antiphago-
cytosis by inhibiting myosin function. Cell Host
Microbe 2, 383–392.
13 Frankel G, Phillips AD, Rosenshine I, Dougan G,
Kaper JB & Knutton S (1998) Enteropathogenic and
enterohaemorrhagic Escherichia coli: more subversive
elements. Mol Microbiol 30, 911–921.
14 Donnenberg M, Yu J & Kaper JB (1993) A second
chromosomal gene necessary for intimate attachment of
enteropathogenic Escherichia coli to epithelial cells.
J Bacteriol 175, 4670–4680.
15 Foubister V, Rosenshine I, Donnenberg MS & Finlay
BB (1994) The eaeB gene of enteropathogenic
Escherichia coli is necessary for signal transduction in
epithelial cells. Infect Immun 62, 3038–3040.
16 Tobe T & Sasakawa C (2002) Species-specific cell adhe-
sion of enteropathogenic Escherichia coli is mediated by
type IV bundle forming pili. Cell Microbiol 4, 29–
42.
D. Hamada et al. EspB as an intrinsically less-ordered effector
FEBS Journal 277 (2010) 2409–2415 ª 2010 The Authors Journal compilation ª 2010 FEBS 2413
17 Taylor KA, Luther PW & Donnenberg MS (1999)
Expression of the EspB protein of enteropathogenic
Escherichia coli within HeLa cells affects stress fibers
and cellular morphology. Infect Immun 67, 120–125.
18 Knutton S, Rosenshine I, Pallen MJ, Nisan L, Neves
BC, Bain C, Wolff C, Dougan G & Frankel G (1998)
A novel EspA-associated surface organelle of entero-
pathogenic Escherichia coli involved in translocation of

EspB into eukaryotic cells. EMBO J 17, 2166–2176.
19 Bajpai S, Feng Y, Krishnamurthy R, Longmore GD &
Wirtz D (2009) Loss of a-catenin decreases the strength
of single E-cadherin bonds between human cancer cells.
J Biol Chem 284, 18252–18259.
20 Zarivach R, Vuckovic M, Deng W, Finlay B &
Strynadka NC (2007) Structural analysis of a prototypi-
cal ATPase from the type III secretion system. Nat
Struct Mol Biol 14, 131–137.
21 Zaribach R, Deng W, Vuckovic M, Felise HB, Nguyen
HV, Miller SI, Finlay BB & Strynadka NC (2008)
Structural analysis of the essential self-cleaving type III
secretion proteins EscU and SpaS. Nature 453, 124–127.
22 Luo Y, Frey EA, Pfuetzner RA, Creagh AL, Knoechel
DG, Haynes CA, Finlay BB & Strynadka NC (2000)
Crystal structure of enteropathogenic Escherichia coli
intimin–receptor complex. Nature 405, 1073–1077.
23 Luo Y, Bertero MG, Frey EA, Pfuetzner RA, Wenk
MR, Creagh L, Marcus SL, Lim D, Sicheri F, Kay C
et al. (2001) Structural and biochemical characterization
of the type III secretion chaperones CesT and SigE. Nat
Struct Biol 8, 1031–1036.
24 Yip CK, Kimbrough TG, Felise HB, Vuckovic M,
Thomas NA, Pfuetzner RA, Frey EA, Finlay BB,
Miller SI & Strynadka NC (2005) Structural character-
ization of the molecular platform for type III secretion
system assembly. Nature 435, 702–707.
25 Yip CK, Finlay BB & Strynadka NC (2005) Structural
characterization of a type III secretion filament protein
in complex with its chaperone. Nat Struct Mol Biol 12,

75–81.
26 Jobichen C, Li M, Yerushalmi G, Tan YW, Mok YK,
Rosenshine I, Leung KY & Sivaraman J (2007) Structure
of GriR and the implication of its EDED motif in medi-
ating the regulation of type III secretion system in EHEC.
PLoS Pathog 3, e69, doi:10.1371/journal.ppat.0030069.
27 Hsu Y, Jubelin G, Taieb F, Nougayre
´
de JP, Oswald E
& Stebbins CE (2008) Structure of the cyclomodulin Cif
from pathogenic Escherichia coli. J Mol Biol 384, 465–
477.
28 Spreter T, Yip CK, Sanowar S, Andre I, Kimbrough
TG, Vuckovic M, Pfuetzner RA, Deng W, Yu AC,
Finlay BB et al. (2009) A conserved structural motif
mediates formation of the periplasmic rings in the type
III secretion system. Nat Struct Mol Biol 16, 468–476.
29 Kelly G, Prasannan S, Daniell S, Fleming K, Frankel
G, Dougan G, Connerton I & Matthews S (1999)
Structure of the cell-adhesion fragment of intimin from
enteropathogenic Escherichia coli. Nat Struct Biol 6,
313–318.
30 Batchelor M, Prasannan S, Daniell S, Reece S, Conner-
ton I, Bloomberg G, Dougan G, Frankel G &
Matthews S (2000) Structural basis for recognition of
the translocated intimin receptor (Tir) by intimin from
enteropathogenic Escherichia coli. EMBO J 19
, 2452–
2464.
31 Cheng HC, Skehan BM, Campellone KG, Leong JM &

Rosen MK (2008) Structural mechanism of WASP acti-
vation by the enterohaemorrhagic E. coli effector
EspFU (2008). Nature 454, 1009–1013.
32 Wang YA, Yu X, Yip C, Strynadka NC & Egelman
EH (2006) Structural polymorphism in bacterial EspA
filaments revealed by cryo-EM and an improved
approach to helical reconstruction. Structure 14, 1189–
1196.
33 Dunker AK, Lawson JD, Brown CJ, Williams RM,
Romero P, Oh JS, Oldfield CJ, Campen AM, Ratiff
CM, Hipps Z et al. (2001) Intrinsically disordered
protein. J Mol Graph Model 19, 26–59.
34 Dyson HJ & Wright PE (2005) Intrinsically unstruc-
tured proteins and their functions. Nat Rev Mol Cell
Biol 6, 197–208.
35 Ohgushi M & Wada A (1983) ‘Molten-globule state’: a
compact form of globular proteins with mobile side-
chains. FEBS Lett 164, 21–24.
36 Arai M & Kuwajima K (2000) Role of the molten glob-
ule state in protein folding. Adv Protein Chem 53, 209–
282.
37 Faudry E, Job V, Dessen A, Attree I & Forge V (2007)
Type III secretion system translocator has a molten
globule conformation both in its free and chaperone-
bound forms. FEBS J 274, 3601–3610.
38 Li X, Romero P, Rani M, Dunker AK & Obradovic Z
(1999) Predicting protein disorder for N-, C- and inter-
nal regions. Genome Inform 10, 30–40.
39 Romero P, Obradovic Z, Li X, Garner E, Brown C &
Dunker AK (2001) Sequence complexity of disordered

protein. Proteins 42, 38–48.
40 Romero P, Obradovic Z & Dunker AK (1997) Sequence
data analysis for long disordered regions prediction in
the calcineurin family. Genome Inform 8, 110–124.
41 Mounier J, Popoff MR, Enninga J, Frame MC, Sanso-
netti PJ & van Nhieu GT (2009) The IpaC carboxyter-
minal effector domain mediates Src-dependent actin
polymerization during Shigella invasion of epithelial
cells. PLoS Pathog 5, e1000271.
42 van Nhieu TG, Bourdet-Sicard R, Dume
´
nil G, Blocker
A & Sansonetti PJ (2000) Bacterial signals and cell
responses during Shigella entry into epithelial cells. Cell
Microbiol 2, 187–193.
43 Bourdet-Sicard R, Egille C, Sansonett PJ & van Nhieu
GT (2000) Diversion of cytoskeletal processes by
EspB as an intrinsically less-ordered effector D. Hamada et al.
2414 FEBS Journal 277 (2010) 2409–2415 ª 2010 The Authors Journal compilation ª 2010 FEBS
Shigella during invasion of epithelial cells. Microbes
Infect 2, 813–819.
44 Hayward RD & Koronakis V (1999) Direct nucleation
and bundling of actin by the SipC protein of invasive
Salmonella. EMBO J 18, 4926–4934.
45 Faudry E, Vernier G, Neumann E, Forge V & Attree I
(2006) Synergistic pore formation by type III toxin
translocators of Pseudomonas aeruginosa. Biochemistry
45, 8117–8123.
46 Tardy F, Homble
´

F, Neyt C, Wattiez R, Comelis
GR, Ruysschaert J-M & Cabiaux V (1999)
Yersinia enterocolitica type III secretion–translocation
system: channel formation by secreted Yops.
EMBO J 18, 6793–6799.
47 Kueltzo LA, Osiecki J, Barker J, Picking WL, Ersoy B,
Picking WD & Middaugh CR (2003) Structure–function
analysis of invasion plasmid antigen C (IpaC) from
Shigella flexneri. J Biol Chem 278, 2792–2798.
48 Raab R & Swietnicki W (2003) Yersinia pestis YopD
150–287 fragment is partially unfolded in the native
state. Protein Expr Purif 58, 53–60.
49 von Pawel-Rammingen U, Telepnev MV, Schmidt G,
Aktories K, Walf-Watz H & Rosqbist R (2000) GAP
activity of the Yersinia YopE cytotoxin specifically tar-
gets the Rho pathway: a mechanism for disruption of
actin microfilament structure. Mol Microbiol 36, 737–
748.
50 Evdokimov AG, Tropea JE, Routzahn KM & Waugh
DS (2002) Crystal structure of the Yersinia pestis
GTPase activator YopE. Protein Sci 11, 401–408.
51 Rodgers L, Gamez A, Riek R & Ghosh P (2008) The
type III secretion chaperone SycE promotes a localized
disorder-to-order transition in the natively unfolded
effector YopE. J Biol Chem 283, 20857–20863.
52 Race PR, Solovyova AS & Banfield MJ (2007) Confor-
mation of the EPEC Tir protein in solution: investigat-
ing the impact of serine phosphorylation at positions
434 ⁄ 463. Biophys J 93 , 586–596.
53 Namba K (2001) Roles of partly unfolded conforma-

tions in macromolecular self-assembly. Genes Cells 6,
1–12.
54 Bienkiewicz EA, Adkins JN & Lumb KJ (2002) Func-
tional consequences of preorganized helical structure in
the intrinsically disordered cell-cycle inhibitor
p27(Kip1). Biochemistry 41, 752–759.
55 He
´
brard E, Bessin Y, Michon T, Longhi S, Uversky
VN, Delalande F, van Dorsselar A, Romero P, Walter
J, Declerk N et al. (2009) Intrinsic disorder in viral
proteins genome-linked: experimental and predictive
analyses. Virol J 6, 23.
D. Hamada et al. EspB as an intrinsically less-ordered effector
FEBS Journal 277 (2010) 2409–2415 ª 2010 The Authors Journal compilation ª 2010 FEBS 2415