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
Trang 1Cytoskeleton-modulating effectors of enteropathogenic and enterohemorrhagic Escherichia coli: a case for EspB as
an intrinsically less-ordered effector
Daizo Hamada1, Mitsuhide Hamaguchi2, Kayo N Suzuki3, Ikuhiro Sakata2and Itaru Yanagihara4
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], EspFU [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: daizo@med.kobe-u.ac.jp
(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.
Trang 2to the multifunctionality of this protein [10] EspB
binds to various host proteins including a-catenin [2],
a1-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⁄ 3 in 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 producinitia-tion 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.
Trang 3induced 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 15N–1H
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 stintermedi-ate that is accumulintermedi-ated 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; http://www.pondr.com) 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 a1-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
2 dmol
250 230 210
190
10
130
120
110
Fig 2 Structural properties of EspB Far-UV CD and1H-15N 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.
Trang 4of 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 EspFU⁄ Wiskott–Aldrich syndrome protein complex through either the insulin receptor tyrosine kinase 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 algorithm also predicts that large regions of Tir and EspFU 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
1.0
0.5
0.0
1.0
0.5
0.0
1.0
0.5
0.0
300 200 100
0
Residue number
300 200 100
0
Residue number
300 200
100
0
Residue number
400 300 200
100
0
Residue number
EspB
YopD
EspFu
α1-Antitrypsin
EPEC
EHEC
1.0
0.5
0.0
1.0
0.5
0.0
400 300 200 100 0 Residue number
400 300 200 100 0 Residue number
IpaC
SipC
1.0 0.8 0.6 0.4 0.2 0.0
1.0 0.8 0.6 0.4 0.2 0.0
600 400 200 0 Residue number Tir
200 150 100 50 0 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 EspFU
and Tir from EHEC The predictions derived from PONDR[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 a1-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].
Trang 5We 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,
EspFU 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)
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