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: 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. 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 ; 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 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. 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