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Shigella type III secretion effectors: how, where, when, for what purposes?
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Shigella type III secretion effectors: how, where, when, for what purposes? Claude Parsot1,2 Bacteria of Shigella spp., the causative agents of shigellosis in humans, possess a repertoire of 25–30 effectors injected into host cells by a type III secretion apparatus (T3SA). The T3SA activity is activated upon contact of bacteria with cells and controls expression of some effectors. Recent structural and functional studies suggest that two different sets of effectors are involved in inducing actin cytoskeleton reorganization to promote entry of bacteria into epithelial cells and in modulating cell signaling pathways to dampen innate immune responses induced upon infection, respectively. Schematically, effectors involved in entry are produced independently of the T3SA activity, whereas effectors involved in controlling the cell responses are produced upon activation of the T3SA. Addresses 1 Institut Pasteur, Unite´ de Pathoge´nie Microbienne Mole´culaire, 25 rue du Dr Roux, 75724 Paris Cedex 15, France 2 INSERM U786, 25 rue du Dr Roux, 75724 Paris Cedex 15, France Corresponding author: Parsot, Claude ([email protected])
Current Opinion in Microbiology 2009, 12:110–116 This review comes from a themed issue on Host-microbe interactions: Bacteria Edited by Brendan Kenny and Raphael Valdivia Available online 20th January 2009 1369-5274/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2008.12.002
Introduction Quis, quid, ubi, quibus auxiliis, cur, quomodo, quando Marcus Fabius Quintilianus (ca. 35—ca. 100) The genus Shigella comprises four species, S. boydii, S. dysenteriae, S. flexneri and S. sonnei. Shigellosis in humans is characterized by the destruction of the colonic epithelium provoked by the inflammatory response that is induced upon invasion of the mucosa by bacteria. Shigella enters epithelial cells by promoting membrane ruffling, disrupts the membrane of the vacuole in which it is internalized, multiplies within the cell cytoplasm and induces actin polymerization at one of its poles, which promotes the movement of the bacterium and leads to the formation of a protrusion containing a bacterium at its tips and penetrating into an adjacent cell, disrupts the two cell membranes Current Opinion in Microbiology 2009, 12:110–116
surrounding the bacterium in the protrusion and disseminates from cell to cell. Following its internalization by macrophages, Shigella disrupts the membrane of the phagosome and triggers a pro-inflammatory type of apoptosis. An 220-kb plasmid encodes, in particular, a type III secretion (T3S) system required to trigger entry into epithelial cells and apoptosis in macrophages [1–3]. Other proteins involved in pathogenicity, such as the serine proteases SepA, Pic and SigA and the outermembrane VirG/IcsA involved in the movement of intracellular bacteria, are secreted or presented at the bacterial surface by a C-terminal autotransporter domain [4,5]. As yet, there are no functional studies on the possible involvement of other specialized secretion pathways in pathogenicity. This review focuses on T3S effectors: when are they expressed and delivered to host cells, what are their catalytic activities and their substrates or their interaction partners and in what mechanisms are they involved?
The Shigella T3S system The T3S system is composed of 50 proteins, including Mxi and Spa proteins involved in assembly and regulation of the T3SA, chaperones (IpgA, IpgC, IpgE and Spa15), transcription activators (VirF, VirB and MxiE), translocators (IpaB, IpaC and IpaD) and 25 effectors (Table 1), all of which are encoded by the virulence plasmid, and 5– 7 effectors of the IpaH family encoded by the chromosome. Production of MxiE and the T3SA component Spa13 is dependent upon transcriptional slippage [6,7]. Genes encoding all these proteins exhibit a similar G + C content (34% G + C), suggesting that the whole system was acquired en bloc by lateral transfer [8]. Homologues of Shigella effectors are encoded by a number of other bacteria (Table 2). During growth of bacteria in broth at 37 8C, the T3SA is assembled but is not active and translocators and some effectors are stored in association with dedicated chaperones. The T3SA activity is activated upon contact with host cells (Box 1), which promotes transit of translocators and stored effectors [9] and induces transcription of genes encoding a second set of effectors under the control of MxiE and IpgC, the later acting as a co-activator [10]. Before T3SA activation, IpgC is associated with and act as a chaperone for IpaB and IpaC, while MxiE is associated with the T3SA substrate OspD1 acting as an anti-activator [11]. Following secretion of IpaB, IpaC and OspD1, an interaction between MxiE and IpgC is proposed to allow MxiE to activate transcription of genes and operons containing the 17-bp MxiE box in their promoter region [12]. www.sciencedirect.com
Shigella type III secretion effectors: how, where, when, for what purposes? Parsot 111
Table 1 Features of Shigella effectors. Effector
Size a
IpaB IpaC IpaD IpaA IpaJ IpgD IpgB1 IpgB2 IcsB OspC2 OspC3 OspD1 OspZ OspD2 OspB OspC1 OspF VirA OspD3 OspE1 & 2 OspG IpaH9.8 IpaH7.8 IpaH1.4 IpaH4.5 IpaH2.5 IpaH3 i
580 363 322 633 259 538 208 188 494 484 484 225 230 569 288 470 239 401 565 88 196 545 565 575 574 563 571
T3SA b
Chaperone c
Struct. d
Activity e
Substrate f
IpgC IpgC
Interaction partner g CD44, a5b1, Caspase, Mad2L2
+ Spa15
Vinculin
IpgE Spa15 Spa15 IpgA Spa15 Spa15 Spa15
/+ /+ /+ /+
Phosphatase
PtdIns-(4,5)P2 ELMO1/2 CRICK, ROCKI, ROCKII, mDia1 VirG (bacterial protein)
Spa15 + +
+ + + + + + +
p-Thr lyase
p-p38 & p-Erk1/2 a & b tubulins
Protein kinase Ub ligase Ub ligase Ub ligase
UbcH5, UbcH7, Ste7 (yeast) Ste7 (yeast)
h
+
+
Ub ligase
a
Number of residues. Signs , +/ and + indicate that expression of the corresponding gene is not, partially or fully controlled by the T3SA activity. c T3S chaperone associated with the effector in the bacterial cytoplasm. d The sign + indicates that the structure of the effector has been elucidated. e Enzymatic activity. f Identified substrates for the enzymatic activity. g Partner(s) of interaction identified in the cell. h The presence of an insertion sequence immediately upstream from ipaH2.5 suggests that ipaH2.5 is not expressed. i Other IpaH proteins encoded by the chromosome are not indicated. b
Box 1 When is the T3SA active? The T3SA is not active when bacteria are growing in broth and is activated within seconds upon contact of bacteria with host cells [9]. Determining the relative activity of the T3SA in intracellular bacteria is challenging; lacZ transcriptional fusions driven by MxiE-dependent virA and ipaH promoters were induced upon entry of bacteria within cells and were no longer transcribed in bacteria growing in the intracellular environment, i.e. between 1 and 3 h after entry. By contrast, an ipgD–lacZ fusion, the transcription of which is not dependent upon MxiE, was still expressed by intracellular bacteria [48]. IpaB, IpaC and the T3SA are required for disruption of the cell membranes surrounding bacteria in protrusions during cell-to-cell spread [49,50]. Together, these observations suggest that the T3SA is activated upon contact of bacteria with host cells, deactivated when bacteria multiply in the cell cytoplasm and reactivated when bacteria are in protrusions. However, some reports indicate that, IpaB was secreted by bacteria for more than 1 h after infection of macrophages [51] and that, in epithelial cells infected for 3 h, VirA and IcsB were involved in the movement of intracellular bacteria and their escape from autophagy, respectively [22,32].
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Effectors were classified into three categories (Figure 1), depending on whether their expression is not, partially or fully controlled by the T3SA activity [13]; they are presented below in that order, focusing on those for which cellular binding partners and/or catalytic activities have been recently characterized.
Effectors produced independently of the T3SA activity In addition to their role as translocators, IpaB and IpaC are endowed with effector-like activities. Purified IpaC induced actin polymerization and the formation of filopodial and lamellipodial extensions that were dependent upon Cdc42 and Rac in semi-permeabilized cells and IpaB was shown to interact with CD44, the integrin a5b1 and caspase-1 (reviewed in reference [2]). The N-terminal domain of IpaB was recently shown to interact with the anaphase-promoting complex/cyclosome inhibitor Mad2L2 in vitro and in transfected cells and to modulate the cell cycle progression; this interaction was proposed to slow down cell renewal during infection, thereby Current Opinion in Microbiology 2009, 12:110–116
112 Host-microbe interactions: Bacteria
Table 2 Homologues of Shigella effectors encoded by other bacteriaa. Effector
Salm
Burk
Chro
Soda
Prov
IpaB IpaC IpaA IpaD IpgD OspCs OspF IpaHs IpaJ IcsB OspB OspZ OspG IpgBs VirA OspDs OspE
SipB SipC SipA SipD SopB + SpvC SspHs +
BipB BipC
+
+
BipD
+ + + +
+ +
VirA
+ VirA
+ + +
EPEC
Citr
Yers
+
Pseu
HopAI1 +
BopA + + +
+ NleE + Map/EspM EspG + +
NleE + EspM EspG
+
+
a Bacteria encoding >1 protein exhibiting >25% sequence identity over >50% of the length of a Shigella effector by BLASTp analysis are indicated. The gene encoding the corresponding effector is not necessarily present in all species, serovars or strains; Burk, Burkholderia oklahomensis, thailandensis and/or pseudomallei; Chro, Chromobacterium violaceum; Citr, Citrobacter rodentium; EPEC, Escherichia coli O157:H7; Prov, Providencia alcalifaciens, rustigiani and/or stuartii; Pseu, Pseudomonas syringae pv. Tomato; Salm, Salomonella enterica and/or cholerasuis; Soda, Sodalis glossinidus; Yers, Yersinia pseudotuberculosis and/or pestis.
prolonging the bacterial colonization of the intestinal epithelium. However, a strain producing an IpaB variant that did not interact with Mad2L2, but was still able to promote entry of bacteria, exhibited a competition index of only 0.7 upon co-infection of rabbit ligated ileal loops with the wild-type strain [14]; this experimental model might not be sensitive enough to confirm the role of the IpaB–Mad2L2 interaction during infection. Figure 1
Differential expression of effectors by the T3SA activity. Effectors produced independently of the T3SA activity are indicated in the green box, effectors whose production is positively controlled by the T3SA activity are indicated in the purple box and effectors the transcription of which was detected when the T3SA is not active and increased when the T3SA is active are shown in the overlap [13]. Production of components of the T3SA, chaperones and MxiE (not shown) is not controlled by the T3SA activity and production of IpaH proteins encoded by the chromosome (not shown) is controlled by the T3SA activity [10]. Effectors involved in entry are indicated in blue and effectors involved in dampening the host innate responses are indicated in red. IpaB, IpaC and IpaD (underlined) are the proposed translocators essential for delivery of effectors within host cells. Current Opinion in Microbiology 2009, 12:110–116
IpaA associates with vinculin. Each of the two vinculin binding sites located in the IpaA C-terminal domain (74 residues) consists in an amphipatic a-helix that can insert between helices a1 and a2 of vinculin, thereby stabilizing the open conformation of vinculin [15,16]. Binding of IpaA to the vinculin head domain promoted association of the IpaA:vinculin complex to F-actin and depolymerization of actin filaments [17]. It is unlikely that only the C-terminal domain of IpaA is endowed with a function; in transfected cells, production of IpaA containing, or not, the vinculin binding domain led to increased Rho activity independently of vinculin and the IpaA vinculin binding domain [18]. IpgD dephosphorylates phosphatidylinositol 4,5-biphosphate (PtdIns-(4,5)P2) into phosphatidylinositol 5-monophophate (PtdIns-(5)P) in vitro and upon infection of epithelial cells [19]. Reducing the amount of PtdIns(4,5)P2 in the plasma membrane decreases the adhesion energy of the cortical cytoskeleton to the membrane, which probably facilitates extension of membrane ruffles during entry, consistent with the observation that an ipgD mutant provoked less membrane ruffling on epithelial cells [20]. Production of PtdIns-(5)P by IpgD upon infection of epithelial cells also led to activation of the PI3kinase pathway, promoting phosphorylation of Akt and the Akt substrates GSK3 and FKHR and enhancing survival of the host cell [21]. IcsB was shown to bind in vitro to the outermembrane protein VirG/IcsA involved in inducing actin nucleation at one pole of intracellular bacteria. Association of an icsB mutant with autophagosomes was detected in cells www.sciencedirect.com
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infected for 4–6 h. By masking a binding site for the autophagy protein Atg5 onto VirG, binding of IcsB to VirG was proposed to prevent bacteria to be trapped in autophagosomes [22]. The binding of IcsB to VirG is intriguing, because the difference in G + C content of icsB and virG suggests that these genes were acquired from different sources. Inactivation of ipgB1 or ipgB2 had little effect on the ability of bacteria to enter epithelial cells; an ipgB1 ipgB2 mutant was less invasive than the wild-type strain and exhibited an attenuated virulence upon infection of eyes in Guinea pigs and lungs in mice [23,24]. In transfected cells, IpgB2 induced formation actin stress fibres similar to those induced by activation of RhoA, but independently of RhoA. IpgB2 was shown to interact with the Rho binding domain of CRIK, ROCKI, ROCKII and mDia1. In transfected cells, IpgB1 induced the formation of membrane ruffles similar to those induced by activation of Rac1. These observations and a detailed analysis of the effects of Map, an E. coli homologue of IpgB1 and IpgB2, suggested that these effectors act as functional mimics of activated GTPases of the Ras-like family to modulate actin dynamics in the cell; these proteins contain an invariant motif (WxxxE) essential for their activity [25]. IpgB1 was shown to bind ELMO1 and ELMO2 and to mimic the role of activated RhoG by inducing the membrane localization of the ELMO:Dock180 complex and the subsequent activation of Rac1 [26]. OspD1, the MxiE anti-activator [11], is also a potential effector. Expression of IpaJ in Saccharomyces cerevisiae induced a strong growth inhibition, which led to the identification of IpaJ as T3SA substrate [27]. The function of OspC2, OspC3 and OspD2 is not known. OspZ has 230 residues in S. dysenteriae, S. sonnei and S. boydii, but, owing to a frameshift mutation only 188 residues in S. flexneri. Inactivation of ospZ in S. flexneri had no effect on the ability of bacteria to enter into and disseminate within epithelial cells or elicit keratoconjunctivitis in Guinea pigs, but impaired induction of transmigration of polymorphonuclear cells through a polarized monolayer of infected epithelial cells [28].
Effectors whose expression is increased by T3SA activation These proteins correspond to OspB, OspC1, OspF and VirA. There is not much information on the function of OspB [29] and OspC1 [30]. OspF is a phosphothreonine lyase that irreversibly inactivates the mitogen-activated protein kinases (MAPK) p-p38 and p-Erk1/2 by removing the phosphate group from the phosphothreonine of the MAPK activation loop [35]. Infection of epithelial cells by an ospF mutant, but not the wild-type strain, led to www.sciencedirect.com
accumulation of p-Erk and p-p38 [36,37] and an increased phosphorylation on Ser10 of histone H3; the lack of phosphorylation of this histone in cells infected by the wild-type strain, as a consequence of MAPK inactivation by OspF, was proposed to be responsible for the lack of induction of a set of NF-kB-controlled genes [36]. Upon infection of ligated ileal loops in rabbits [36] and lungs in mice [37], ospF mutants caused a more severe mucosal destruction and induced an increased recruitment of polymorphonuclear cells than the wild-type strain. Determination of the structures of wild-type SpvC (the Salmonella enterica OspF homologue) and a SpvC inactive variant in complex with a phosphorylated peptide and a detailed enzymatic analysis shed light on the activity of OspF/SpvC family members and explained their substrate specificity towards p-p38 [38]. The N-terminal region of OspF (residues 1–24) carrying the secretion signal contains a motif similar to the one involved in the docking of cellular proteins to MAPKs; this motif was shown to be necessary and sufficient for the interaction of OspF with Erk2 [38]. VirA was shown to interact with a-b tubulin heterodimers and to induce microtubule destabilization, leading to activation of Rac1 and promoting membrane ruffling in infected cells [31]. VirA secreted by intracellular bacteria was shown to facilitate the movement of bacteria in the cell cytoplasm owing to its cysteine protease activity towards a-tubulin [32]. However, the structure of VirA does not exhibit any similarity to papain-like cysteine proteases, and the role of purified VirA in the degradation of tubulin or disassembly of microtubules has not been confirmed [33,34].
Effectors produced in response to T3SA activation Effectors whose production is dependent upon MxiE and the T3SA activity include OspD3/SenA, OspE1, OspE2, OspG and members of the IpaH family. Inactivation of mxiE did not affect the ability of bacteria to enter epithelial cells, suggesting that these effectors are not required for entry [10]. Unexpectedly for a T3SA substrate, secreted OspD3/SenA, i.e. present in the culture medium, was shown to be endowed with an enterotoxin activity [39]. OspE proteins are encoded by two almost identical genes (ospE1 and ospE2) in S. flexneri. In S. sonnei, ospE1 carries a frameshift mutation and inactivation of ospE2 did not affect the ability of bacteria to enter epithelial cells but led to the formation of smaller plaques on a confluent cell monolayer. Cells infected for 3 h by the ospE2 mutant, but not the wild-type strain, exhibited an altered morphology characterized by a rounding shape. A HAtagged OspE2 protein produced by S. sonnei accumulated at focal contact-like structures in infected cells [40]. Current Opinion in Microbiology 2009, 12:110–116
114 Host-microbe interactions: Bacteria
OspG exhibits sequence similarities with protein kinases and was shown to be endowed with an autophosphorylation activity and to interact with ubiquitinated E2 ubiquitin conjugating enzymes (Ubc), including UbcH5 and UbcH7. In transfected cells, production of OspG prevented degradation of IkBa induced by TNF-a, suggesting that OspG interferes with ubiquitination of p-IkBa by the E3 ubiquitin ligase SCFb-TrCP. As compared with the wild-type strain, an ospG mutant induced a faster degradation of p-IkBa in infected cells and a stronger inflammatory response in rabbit ligated ileal loops [41]. IpaH proteins are characterized by a variable N-terminal domain containing six to eight Leucine-rich repeats (LRR) and a conserved C-terminal domain of 300 residues. In yeast, production of IpaH9.8 interrupted the pheromone response pathway by inducing the proteasome-dependent degradation of the MAPK kinase Ste7 and, in vitro, IpaH9.8 was shown to be endowed with an E3 ubiquitin ligase activity towards Ste7 [42]. The catalytic activity of the IpaH C-terminal domain is dependent upon a Cys residue involved in the transfer of ubiquitin to the substrate through a transthiolation reaction, as described for HECT domain containing E3s; this domain does not exhibit any structural similarities with eukaryotic E3s [43,44]. Differences in the substrate specificity of IpaH9.8, IpaH1.4 and SspH1 (a Salmonella enterica IpaH homologue) suggested that the LRR-containing N-terminal domain corresponds to the substrate recognition domain [42,43]. Intriguingly, the C-terminal domain of IpaH9.8 was shown to interact with the splicing factor U2AF35 [45]. Following intranasal infection of mice, an ipaH9.8 mutant and a mutant in which all chromosomal ipaH genes were inactivated provoked a stronger inflammatory response and a reduced bacterial colonization than the wild-type strain [45,46].
bacteria do not enter, rather than a post-invasion stage, i.e. in infected cells. Remarkable progresses have been made in elucidating the structure and deciphering the function of a number of Shigella effectors. Once a catalytic activity, or a binding partner, has been identified, there might be a long way between the characterization of a substrate and the identification of the substrate whose modification has been selected throughout evolution as beneficial to the pathogen. Even though generalizations must be taken cautiously, trends seem to emerge; some effectors promote entry of bacteria within epithelial cells, whereas others dampen innate immune responses induced upon infection. Interestingly, effectors involved in entry are produced independently of the T3SA activity, whereas effectors involved in controlling cell signaling are produced in response to activation of the T3SA. Unraveling the functions of effectors points to the how and the for what purposes; we still have much to learn on the where and the when effectors are effective.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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7.
Penno C, Hachani A, Biskri L, Sansonetti P, Allaoui A, Parsot C: Transcriptional slippage controls production of type III secretion apparatus components in Shigella flexneri. Mol Microbiol 2006, 62:1460-1468.
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Concluding remarks Control of effector-encoding genes by the T3SA activity imposes a hierarchy in the delivery of effectors to target cells; effectors produced before activation of the T3SA should transit before effectors produced in response to activation of the T3SA. However, except for effectors involved in entry, we do not know the stage of the infection at which effectors are most needed; is it when bacteria are within epithelial cells, or when they are facing inflammatory cells recruited to the site of infection, or at the onset of infection to promote bacterial colonization and survival? MxiE-controlled effectors delivered at the apical side of epithelial cells by presumably extracellular bacteria were shown to suppress transcription of genes encoding antimicrobial peptides and chemokines and, upon infection of human intestinal xenotransplants in mice, to promote progression of bacteria towards intestinal crypts [47]. Thus, MxiE-controlled effectors might be involved at a pre-invasion stage, i.e. in cells in which Current Opinion in Microbiology 2009, 12:110–116
9.
Enninga J, Mounier J, Sansonetti P, Van Nhieu GT: Secretion of type III effectors into host cells in real time. Nat Methods 2005, 2:959-965. This study presents a novel approach to monitor transit of T3SA substrates in real time and shows that, following contact of bacteria with epithelial cells, the T3SA is activated within seconds. www.sciencedirect.com
Shigella type III secretion effectors: how, where, when, for what purposes? Parsot 115
10. Mavris M, Page AL, Tournebize R, Demers B, Sansonetti P, Parsot C: Regulation of transcription by the activity of the Shigella flexneri type III secretion apparatus. Mol Microbiol 2002, 43:1543-1553. 11. Parsot C, Ageron E, Penno C, Mavris M, Jamoussi K, d’Hauteville H, Sansonetti P, Demers B: A secreted antiactivator, OspD1, and its chaperone, Spa15, are involved in the control of transcription by the type III secretion apparatus activity in Shigella flexneri. Mol Microbiol 2005, 56:1627-1635. 12. Mavris M, Sansonetti PJ, Parsot C: Identification of the cisacting site involved in activation of promoters regulated by activity of the type III secretion apparatus in Shigella flexneri. J Bacteriol 2002, 184:6751-6759. 13. Le Gall T, Mavris M, Martino MC, Bernardini ML, Denamur E, Parsot C: Analysis of virulence plasmid gene expression defines three classes of effectors in the type III secretion system of Shigella flexneri. Microbiology 2005, 151:951-962. 14. Iwai H, Kim M, Yoshikawa Y, Ashida H, Ogawa M, Fujita Y, Muller D, Kirikae T, Jackson PK, Kotani S et al.: A bacterial effector targets Mad2L2, an APC inhibitor, to modulate host cell cycling. Cell 2007, 130:611-623. 15. Hamiaux C, van Eerde A, Parsot C, Broos J, Dijkstra BW: Structural mimicry for vinculin activation by IpaA, a virulence factor of Shigella flexneri. EMBO Rep 2006, 7:794-799. 16. Izard T, Van Nhieu GT, Bois PRJ: Shigella applies molecular mimicry to subvert vinculin and invade host cells. J Cell Biol 2006, 175:465-475. 17. Ramarao N, Le Clainche C, Izard T, Bourdet-Sicard R, Ageron E, Sansonetti PJ, Carlier MF, Tran Van Nhieu G: Capping of actin filaments by vinculin activated by the Shigella IpaA carboxylterminal domain. FEBS Lett 2007, 581:853-857. 18. DeMali KA, Jue AL, Burridge K: IpaA targets beta 1 integrins and Rho to promote actin cytoskeleton rearrangements necessary for Shigella entry. J Biol Chem 2006, 281:39534-39541. 19. Niebuhr K, Giuriato S, Pedron T, Philpott DJ, Gaits F, Sable J, Sheetz MP, Parsot C, Sansonetti PJ, Payrastre B: Conversion of PtdIns(4,5)P-2 into PtdIns(5)P by the S. flexneri effector IpgD reorganizes host cell morphology. EMBO J 2002, 21:5069-5078. 20. Niebuhr K, Jouihri N, Allaoui A, Gounon P, Sansonetti PJ, Parsot C: IpgD, a protein secreted by the type III secretion machinery of Shigella flexneri, is chaperoned by IpgE and implicated in entry focus formation. Mol Microbiol 2000, 38:8-19. 21. Pendaries C, Tronchere H, Arbibe L, Mounier J, Gozani O, Cantley L, Fry MJ, Gaits-Iacovoni F, Sansonetti PJ, Payrastre B: PtdIns(5)P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection. EMBO J 2006, 25:1024-1034. 22. Ogawa M, Yoshimori T, Suzuki T, Sagara H, Mizushima N, Sasakawa C: Escape of intracellular Shigella from autophagy. Science 2005, 307:727-731. This study shows that several hours after entry of bacteria into epithelial cells, the effector IcsB is required to prevent intracellular bacteria to be trapped into autophagosomes by masking a binding site for Atg5 onto the outermembrane protein VirG/IcsA involved in the intracellular movement of bacteria (see also Reference [32] for the role of a T3S effector secreted by intracellular bacteria). 23. Ohya K, Handa Y, Ogawa M, Suzuki M, Sasakawa C: IpgB1 is a novel Shigella effector protein involved in bacterial invasion of host cells. J Biol Chem 2005, 280:24022-24034. 24. Hachani A, Biskri L, Rossi G, Marty A, Menard R, Sansonetti P, Parsot C, Van Nhieu GT, Bernardini ML, Allaoui A: IpgB1 and IpgB2, two homologous effectors secreted via the Mxi-Spa type III secretion apparatus, cooperate to mediate polarized cell invasion and inflammatory potential of Shigella flexenri. Microbe Infect 2008, 10:260-268. 25. Alto NM, Shao F, Lazar CS, Brost RL, Chua G, Mattoo S, McMahon SA, Ghosh P, Hughes TR, Boone C et al.: Identification of a bacterial type III effector family with G protein mimicry functions. Cell 2006, 124:133-145. This study based on cells transfected with plasmids encoding members of the IpgB family proposes that these effectors act as functional mimics www.sciencedirect.com
of GTP-active GTPases of the Ras family, IpgB1, IpgB2 and Map functioning as active forms of RhoA, Rac1 and Cdc42, respectively (see also Reference [26]). 26. Handa Y, Suzuki M, Ohya K, Iwai H, Ishijima N, Koleske AJ, Fukui Y, Sasakawa C: Shigella IpgB1 promotes bacterial entry through the ELMO-Dock180 machinery. Nat Cell Biol 2007, 9:U121-U165. This study shows that IpgB1 associates with ELMO and Dock180 to stimulate Rac1 activity and promote membrane ruffling at the site of bacterial entry; IpgB1 is proposed to act as a RhoG mimic (see also Reference [25]). 27. Slagowski NL, Kramer RW, Morrison MF, LaBaer J, Lesser CF: A functional genomic yeast screen to identify pathogenic bacterial proteins. Plos Pathogens 2008:4. By monitoring growth of a collection of yeast expressing virulence plasmid-encoded proteins, the authors propose growth inhibition of yeast as a new assay to identify T3S effectors. 28. Zurawski DV, Mumy KL, Badea L, Prentice JA, Hartland EL, McCormick BA, Maurelli AT: The NleE/OspZ family of effector proteins is required for polymorphonuclear transepithelial migration, a characteristic shared by enteropathogenic Escherichia coli and Shigella flexneri infections. Infect Immun 2008, 76:369-379. 29. Santapaola D, Del Chierico F, Petrucca A, Uzzau S, Casalino M, Colonna B, Sessa R, Berlutti F, Nicoletti M: Apyrase, the product of the virulence plasmid-encoded phoN2 (apy) gene of Shigella flexneri, is necessary for proper unipolar IcsA localization and for efficient intercellular spread. J Bacteriol 2006, 188:1620-1627. 30. Zurawski DV, Mitsuhata C, Mumy KL, McCormick BA, Maurelli AT: OspF and OspC1 are Shigella flexneri type III secretion system effectors that are required for postinvasion aspects of virulence. Infect Immun 2006, 74:5964-5976. 31. Yoshida S, Katayama E, Kuwae A, Mimuro H, Suzuki T, Sasakawa C: Shigella deliver an effector protein to trigger host microtubule destabilization, which promotes Rac1 activity and efficient bacterial internalization. EMBO J 2002, 21:2923-2935. 32. Yoshida S, Handa Y, Suzuki T, Ogawa M, Suzuki M, Tamai A, Abe A, Katayama E, Sasakawa C: Microtubule-severing activity of Shigella is pivotal for intercellular spreading. Science 2006, 314:985-989. 33. Germane KL, Ohi R, Goldberg MB, Spiller BW: Structural and functional studies indicate that Shigella VirA is not a protease and does not directly destabilize microtubules. Biochemistry 2008, 47:10241-10243. 34. Davis J, Wang J, Tropea JE, Zhang D, Daute Z, Waugh DS: Wlodawer A: Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri. Protein Sci 2008, 17:2167-2173. 35. Li HT, Xu H, Zhou Y, Zhang J, Long CZ, Li SQ, Chen S, Zhou JM, Shao F: The phosphothreonine lyase activity of a bacterial type III effector family. Science 2007, 315:1000-1003. This study demonstrates the phosphothreonine lyase activity of OspF that irreversibly inactivates the dually phosphorylated MAPKs Erk1/2, p-38 and JNK in vitro (see also References [36,37] and the follow-up in Reference [38]). 36. Arbibe L, Kim DW, Batsche E, Pedron T, Mateescu B, Muchardt C, Parsot C, Sansonetti PJ: An injected bacterial effector targets chromatin access for transcription factor NF-kappa B to alter transcription of host genes involved in immune responses. Nat Immunol 2007, 8:47-56. 37. Kramer RW, Slagowski NL, Eze NA, Giddings KS, Morrison MF, Siggers KA, Starnbach MN, Lesser CF: Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation. Plos Pathogens 2007, 3:179-190. 38. Zhu YQ, Li HT, Long CZ, Hu LY, Xu H, Liu LP, Chen S, Wang DC, Shao F: Structural insights into the enzymatic mechanism of the pathogenic MAPK phosphothreonine lyase. Mol Cell 2007, 28:899-913. This structural study of wild-type SpvC and an inactive SpvC variant in complex with a phosphopeptide substrate, as well as a detailled enzyCurrent Opinion in Microbiology 2009, 12:110–116
116 Host-microbe interactions: Bacteria
matic analysis, shed light on the reaction mechanism and substrate specificity of OspF and SpvC. 39. Nataro JP, Seriwatana J, Fasano A, Maneval DR, Guers LD, Noriega F, Dubovsky F, Levine MM, Morris JG: Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive Escherichia coli and Shigella strains. Infect Immun 1995, 63:4721-4728. 40. Miura M, Terajima J, Izumiya H, Mitobe J, Komano T, Watanabe H: OspE2 of Shigella sonnei is required for the maintenance of cell architecture of bacterium-infected cells. Infect Immun 2006, 74:2587-2595. 41. Kim DW, Lenzen G, Page AL, Legrain P, Sansonetti PJ, Parsot C: The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes. Proc Natl Acad Sci USA 2005, 102:14046-14051. This study presents evidence that OspG is a protein kinase that binds ubiquitinylated ubiquitin-conjugating enzymes and prevents phosphoIkBa degradation induced upon entry of bacteria. 42. Rohde JR, Breitkreutz A, Chenal A, Sansonetti PJ, Parsot C: Type III secretion effectors of the IpaH family are E3 ubiquitin ligases. Cell Host Microbe 2007, 1:77-83. This study assigns a function for IpaH family members as E3 ubiquitin ligases and suggests that the variable N-terminal domain confers substrate specificity and the conserved C-terminal domain carries the catalytic activity (see the follow-up in References [43,44]). 43. Singer AU, Rohde JR, Lam R, Skarina T, Kagan O, Dileo R, Chirgadze NY, Cuff ME, Joachimiak A, Tyers M et al.: Structure of the Shigella T3SS effector IpaH defines a new class of E3 ubiquitin ligases. Nat Struct Mol Biol 2008, 15:1293-1301. 44. Zhu Y, Li H, Hu L, Wang J, Zhou Y, Pang Z, Liu L, Shao F: Structure of a Shigella effector reveals a new class of ubiquitin ligases. Nat Struct Mol Biol 2008, 15:1302-1308. 45. Okuda J, Toyotome T, Kataoka N, Ohno M, Abe H, Shimura Y, Seyedarabi A, Pickersgill R, Sasakawa C: Shigella effector
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IpaH9,8 binds to a splicing factor U2AF(35) to modulate host immune responses. Biochem Biophys Res Commun 2005, 333:531-539. 46. Ashida H, Toyotome T, Nagai T, Sasakawa C: Shigella chromosomal IpaH proteins are secreted via the type III secretion system and act as effectors. Mol Microbiol 2007, 63:680-693. 47. Sperandio B, Regnault B, Guo JH, Zhang Z, Stanley SL, Sansonetti PJ, Pedron T: Virulent Shigella flexneri subverts the host innate immune response through manipulation of antimicrobial peptide gene expression. J Exp Med 2008, 205:1121-1132. This study shows that upon infection of polarized human intestinal cells ex vivo and infection of human intestinal xenotransplants in mice, MxiEcontrolled effectors suppress transcription of genes encoding antimicrobial peptides and chemokines and facilitate progression of bacteria towards intestinal crypts. 48. Demers B, Sansonetti PJ, Parsot C: Induction of type III secretion in Shigella flexneri is associated with differential control of transcription of genes encoding secreted proteins. EMBO J 1998, 17:2894-2903. 49. Page AL, Ohayon H, Sansonetti PJ, Parsot C: The secreted IpaB and IpaC invasins and their cytoplasmic chaperone IpgC are required for intercellular dissemination of Shigella flexneri. Cell Microbiol 1999, 1:183-193. 50. Schuch R, Sandlin RC, Maurelli AT: A system for identifying post-invasion functions of invasion genes: requirements for the Mxi-Spa type III secretion pathway of Shigella flexneri in intercellular dissemination. Mol Microbiol 1999, 34:675-689. 51. Schroeder GN, Jann NJ, Hilbi H: Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1dependent macrophage cell death. Microbiol UK 2007, 153:2862-2876.
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Shigella type III secretion effectors: how, where, when, for what purposes? Claude Parsot1,2 Bacteria of Shigella spp., the causative agents of shigellosis in humans, possess a repertoire of 25–30 effectors injected into host cells by a type III secretion apparatus (T3SA). The T3SA activity is activated upon contact of bacteria with cells and controls expression of some effectors. Recent structural and functional studies suggest that two different sets of effectors are involved in inducing actin cytoskeleton reorganization to promote entry of bacteria into epithelial cells and in modulating cell signaling pathways to dampen innate immune responses induced upon infection, respectively. Schematically, effectors involved in entry are produced independently of the T3SA activity, whereas effectors involved in controlling the cell responses are produced upon activation of the T3SA. Addresses 1 Institut Pasteur, Unite´ de Pathoge´nie Microbienne Mole´culaire, 25 rue du Dr Roux, 75724 Paris Cedex 15, France 2 INSERM U786, 25 rue du Dr Roux, 75724 Paris Cedex 15, France Corresponding author: Parsot, Claude ([email protected])
Current Opinion in Microbiology 2009, 12:110–116 This review comes from a themed issue on Host-microbe interactions: Bacteria Edited by Brendan Kenny and Raphael Valdivia Available online 20th January 2009 1369-5274/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2008.12.002
Introduction Quis, quid, ubi, quibus auxiliis, cur, quomodo, quando Marcus Fabius Quintilianus (ca. 35—ca. 100) The genus Shigella comprises four species, S. boydii, S. dysenteriae, S. flexneri and S. sonnei. Shigellosis in humans is characterized by the destruction of the colonic epithelium provoked by the inflammatory response that is induced upon invasion of the mucosa by bacteria. Shigella enters epithelial cells by promoting membrane ruffling, disrupts the membrane of the vacuole in which it is internalized, multiplies within the cell cytoplasm and induces actin polymerization at one of its poles, which promotes the movement of the bacterium and leads to the formation of a protrusion containing a bacterium at its tips and penetrating into an adjacent cell, disrupts the two cell membranes Current Opinion in Microbiology 2009, 12:110–116
surrounding the bacterium in the protrusion and disseminates from cell to cell. Following its internalization by macrophages, Shigella disrupts the membrane of the phagosome and triggers a pro-inflammatory type of apoptosis. An 220-kb plasmid encodes, in particular, a type III secretion (T3S) system required to trigger entry into epithelial cells and apoptosis in macrophages [1–3]. Other proteins involved in pathogenicity, such as the serine proteases SepA, Pic and SigA and the outermembrane VirG/IcsA involved in the movement of intracellular bacteria, are secreted or presented at the bacterial surface by a C-terminal autotransporter domain [4,5]. As yet, there are no functional studies on the possible involvement of other specialized secretion pathways in pathogenicity. This review focuses on T3S effectors: when are they expressed and delivered to host cells, what are their catalytic activities and their substrates or their interaction partners and in what mechanisms are they involved?
The Shigella T3S system The T3S system is composed of 50 proteins, including Mxi and Spa proteins involved in assembly and regulation of the T3SA, chaperones (IpgA, IpgC, IpgE and Spa15), transcription activators (VirF, VirB and MxiE), translocators (IpaB, IpaC and IpaD) and 25 effectors (Table 1), all of which are encoded by the virulence plasmid, and 5– 7 effectors of the IpaH family encoded by the chromosome. Production of MxiE and the T3SA component Spa13 is dependent upon transcriptional slippage [6,7]. Genes encoding all these proteins exhibit a similar G + C content (34% G + C), suggesting that the whole system was acquired en bloc by lateral transfer [8]. Homologues of Shigella effectors are encoded by a number of other bacteria (Table 2). During growth of bacteria in broth at 37 8C, the T3SA is assembled but is not active and translocators and some effectors are stored in association with dedicated chaperones. The T3SA activity is activated upon contact with host cells (Box 1), which promotes transit of translocators and stored effectors [9] and induces transcription of genes encoding a second set of effectors under the control of MxiE and IpgC, the later acting as a co-activator [10]. Before T3SA activation, IpgC is associated with and act as a chaperone for IpaB and IpaC, while MxiE is associated with the T3SA substrate OspD1 acting as an anti-activator [11]. Following secretion of IpaB, IpaC and OspD1, an interaction between MxiE and IpgC is proposed to allow MxiE to activate transcription of genes and operons containing the 17-bp MxiE box in their promoter region [12]. www.sciencedirect.com
Shigella type III secretion effectors: how, where, when, for what purposes? Parsot 111
Table 1 Features of Shigella effectors. Effector
Size a
IpaB IpaC IpaD IpaA IpaJ IpgD IpgB1 IpgB2 IcsB OspC2 OspC3 OspD1 OspZ OspD2 OspB OspC1 OspF VirA OspD3 OspE1 & 2 OspG IpaH9.8 IpaH7.8 IpaH1.4 IpaH4.5 IpaH2.5 IpaH3 i
580 363 322 633 259 538 208 188 494 484 484 225 230 569 288 470 239 401 565 88 196 545 565 575 574 563 571
T3SA b
Chaperone c
Struct. d
Activity e
Substrate f
IpgC IpgC
Interaction partner g CD44, a5b1, Caspase, Mad2L2
+ Spa15
Vinculin
IpgE Spa15 Spa15 IpgA Spa15 Spa15 Spa15
/+ /+ /+ /+
Phosphatase
PtdIns-(4,5)P2 ELMO1/2 CRICK, ROCKI, ROCKII, mDia1 VirG (bacterial protein)
Spa15 + +
+ + + + + + +
p-Thr lyase
p-p38 & p-Erk1/2 a & b tubulins
Protein kinase Ub ligase Ub ligase Ub ligase
UbcH5, UbcH7, Ste7 (yeast) Ste7 (yeast)
h
+
+
Ub ligase
a
Number of residues. Signs , +/ and + indicate that expression of the corresponding gene is not, partially or fully controlled by the T3SA activity. c T3S chaperone associated with the effector in the bacterial cytoplasm. d The sign + indicates that the structure of the effector has been elucidated. e Enzymatic activity. f Identified substrates for the enzymatic activity. g Partner(s) of interaction identified in the cell. h The presence of an insertion sequence immediately upstream from ipaH2.5 suggests that ipaH2.5 is not expressed. i Other IpaH proteins encoded by the chromosome are not indicated. b
Box 1 When is the T3SA active? The T3SA is not active when bacteria are growing in broth and is activated within seconds upon contact of bacteria with host cells [9]. Determining the relative activity of the T3SA in intracellular bacteria is challenging; lacZ transcriptional fusions driven by MxiE-dependent virA and ipaH promoters were induced upon entry of bacteria within cells and were no longer transcribed in bacteria growing in the intracellular environment, i.e. between 1 and 3 h after entry. By contrast, an ipgD–lacZ fusion, the transcription of which is not dependent upon MxiE, was still expressed by intracellular bacteria [48]. IpaB, IpaC and the T3SA are required for disruption of the cell membranes surrounding bacteria in protrusions during cell-to-cell spread [49,50]. Together, these observations suggest that the T3SA is activated upon contact of bacteria with host cells, deactivated when bacteria multiply in the cell cytoplasm and reactivated when bacteria are in protrusions. However, some reports indicate that, IpaB was secreted by bacteria for more than 1 h after infection of macrophages [51] and that, in epithelial cells infected for 3 h, VirA and IcsB were involved in the movement of intracellular bacteria and their escape from autophagy, respectively [22,32].
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Effectors were classified into three categories (Figure 1), depending on whether their expression is not, partially or fully controlled by the T3SA activity [13]; they are presented below in that order, focusing on those for which cellular binding partners and/or catalytic activities have been recently characterized.
Effectors produced independently of the T3SA activity In addition to their role as translocators, IpaB and IpaC are endowed with effector-like activities. Purified IpaC induced actin polymerization and the formation of filopodial and lamellipodial extensions that were dependent upon Cdc42 and Rac in semi-permeabilized cells and IpaB was shown to interact with CD44, the integrin a5b1 and caspase-1 (reviewed in reference [2]). The N-terminal domain of IpaB was recently shown to interact with the anaphase-promoting complex/cyclosome inhibitor Mad2L2 in vitro and in transfected cells and to modulate the cell cycle progression; this interaction was proposed to slow down cell renewal during infection, thereby Current Opinion in Microbiology 2009, 12:110–116
112 Host-microbe interactions: Bacteria
Table 2 Homologues of Shigella effectors encoded by other bacteriaa. Effector
Salm
Burk
Chro
Soda
Prov
IpaB IpaC IpaA IpaD IpgD OspCs OspF IpaHs IpaJ IcsB OspB OspZ OspG IpgBs VirA OspDs OspE
SipB SipC SipA SipD SopB + SpvC SspHs +
BipB BipC
+
+
BipD
+ + + +
+ +
VirA
+ VirA
+ + +
EPEC
Citr
Yers
+
Pseu
HopAI1 +
BopA + + +
+ NleE + Map/EspM EspG + +
NleE + EspM EspG
+
+
a Bacteria encoding >1 protein exhibiting >25% sequence identity over >50% of the length of a Shigella effector by BLASTp analysis are indicated. The gene encoding the corresponding effector is not necessarily present in all species, serovars or strains; Burk, Burkholderia oklahomensis, thailandensis and/or pseudomallei; Chro, Chromobacterium violaceum; Citr, Citrobacter rodentium; EPEC, Escherichia coli O157:H7; Prov, Providencia alcalifaciens, rustigiani and/or stuartii; Pseu, Pseudomonas syringae pv. Tomato; Salm, Salomonella enterica and/or cholerasuis; Soda, Sodalis glossinidus; Yers, Yersinia pseudotuberculosis and/or pestis.
prolonging the bacterial colonization of the intestinal epithelium. However, a strain producing an IpaB variant that did not interact with Mad2L2, but was still able to promote entry of bacteria, exhibited a competition index of only 0.7 upon co-infection of rabbit ligated ileal loops with the wild-type strain [14]; this experimental model might not be sensitive enough to confirm the role of the IpaB–Mad2L2 interaction during infection. Figure 1
Differential expression of effectors by the T3SA activity. Effectors produced independently of the T3SA activity are indicated in the green box, effectors whose production is positively controlled by the T3SA activity are indicated in the purple box and effectors the transcription of which was detected when the T3SA is not active and increased when the T3SA is active are shown in the overlap [13]. Production of components of the T3SA, chaperones and MxiE (not shown) is not controlled by the T3SA activity and production of IpaH proteins encoded by the chromosome (not shown) is controlled by the T3SA activity [10]. Effectors involved in entry are indicated in blue and effectors involved in dampening the host innate responses are indicated in red. IpaB, IpaC and IpaD (underlined) are the proposed translocators essential for delivery of effectors within host cells. Current Opinion in Microbiology 2009, 12:110–116
IpaA associates with vinculin. Each of the two vinculin binding sites located in the IpaA C-terminal domain (74 residues) consists in an amphipatic a-helix that can insert between helices a1 and a2 of vinculin, thereby stabilizing the open conformation of vinculin [15,16]. Binding of IpaA to the vinculin head domain promoted association of the IpaA:vinculin complex to F-actin and depolymerization of actin filaments [17]. It is unlikely that only the C-terminal domain of IpaA is endowed with a function; in transfected cells, production of IpaA containing, or not, the vinculin binding domain led to increased Rho activity independently of vinculin and the IpaA vinculin binding domain [18]. IpgD dephosphorylates phosphatidylinositol 4,5-biphosphate (PtdIns-(4,5)P2) into phosphatidylinositol 5-monophophate (PtdIns-(5)P) in vitro and upon infection of epithelial cells [19]. Reducing the amount of PtdIns(4,5)P2 in the plasma membrane decreases the adhesion energy of the cortical cytoskeleton to the membrane, which probably facilitates extension of membrane ruffles during entry, consistent with the observation that an ipgD mutant provoked less membrane ruffling on epithelial cells [20]. Production of PtdIns-(5)P by IpgD upon infection of epithelial cells also led to activation of the PI3kinase pathway, promoting phosphorylation of Akt and the Akt substrates GSK3 and FKHR and enhancing survival of the host cell [21]. IcsB was shown to bind in vitro to the outermembrane protein VirG/IcsA involved in inducing actin nucleation at one pole of intracellular bacteria. Association of an icsB mutant with autophagosomes was detected in cells www.sciencedirect.com
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infected for 4–6 h. By masking a binding site for the autophagy protein Atg5 onto VirG, binding of IcsB to VirG was proposed to prevent bacteria to be trapped in autophagosomes [22]. The binding of IcsB to VirG is intriguing, because the difference in G + C content of icsB and virG suggests that these genes were acquired from different sources. Inactivation of ipgB1 or ipgB2 had little effect on the ability of bacteria to enter epithelial cells; an ipgB1 ipgB2 mutant was less invasive than the wild-type strain and exhibited an attenuated virulence upon infection of eyes in Guinea pigs and lungs in mice [23,24]. In transfected cells, IpgB2 induced formation actin stress fibres similar to those induced by activation of RhoA, but independently of RhoA. IpgB2 was shown to interact with the Rho binding domain of CRIK, ROCKI, ROCKII and mDia1. In transfected cells, IpgB1 induced the formation of membrane ruffles similar to those induced by activation of Rac1. These observations and a detailed analysis of the effects of Map, an E. coli homologue of IpgB1 and IpgB2, suggested that these effectors act as functional mimics of activated GTPases of the Ras-like family to modulate actin dynamics in the cell; these proteins contain an invariant motif (WxxxE) essential for their activity [25]. IpgB1 was shown to bind ELMO1 and ELMO2 and to mimic the role of activated RhoG by inducing the membrane localization of the ELMO:Dock180 complex and the subsequent activation of Rac1 [26]. OspD1, the MxiE anti-activator [11], is also a potential effector. Expression of IpaJ in Saccharomyces cerevisiae induced a strong growth inhibition, which led to the identification of IpaJ as T3SA substrate [27]. The function of OspC2, OspC3 and OspD2 is not known. OspZ has 230 residues in S. dysenteriae, S. sonnei and S. boydii, but, owing to a frameshift mutation only 188 residues in S. flexneri. Inactivation of ospZ in S. flexneri had no effect on the ability of bacteria to enter into and disseminate within epithelial cells or elicit keratoconjunctivitis in Guinea pigs, but impaired induction of transmigration of polymorphonuclear cells through a polarized monolayer of infected epithelial cells [28].
Effectors whose expression is increased by T3SA activation These proteins correspond to OspB, OspC1, OspF and VirA. There is not much information on the function of OspB [29] and OspC1 [30]. OspF is a phosphothreonine lyase that irreversibly inactivates the mitogen-activated protein kinases (MAPK) p-p38 and p-Erk1/2 by removing the phosphate group from the phosphothreonine of the MAPK activation loop [35]. Infection of epithelial cells by an ospF mutant, but not the wild-type strain, led to www.sciencedirect.com
accumulation of p-Erk and p-p38 [36,37] and an increased phosphorylation on Ser10 of histone H3; the lack of phosphorylation of this histone in cells infected by the wild-type strain, as a consequence of MAPK inactivation by OspF, was proposed to be responsible for the lack of induction of a set of NF-kB-controlled genes [36]. Upon infection of ligated ileal loops in rabbits [36] and lungs in mice [37], ospF mutants caused a more severe mucosal destruction and induced an increased recruitment of polymorphonuclear cells than the wild-type strain. Determination of the structures of wild-type SpvC (the Salmonella enterica OspF homologue) and a SpvC inactive variant in complex with a phosphorylated peptide and a detailed enzymatic analysis shed light on the activity of OspF/SpvC family members and explained their substrate specificity towards p-p38 [38]. The N-terminal region of OspF (residues 1–24) carrying the secretion signal contains a motif similar to the one involved in the docking of cellular proteins to MAPKs; this motif was shown to be necessary and sufficient for the interaction of OspF with Erk2 [38]. VirA was shown to interact with a-b tubulin heterodimers and to induce microtubule destabilization, leading to activation of Rac1 and promoting membrane ruffling in infected cells [31]. VirA secreted by intracellular bacteria was shown to facilitate the movement of bacteria in the cell cytoplasm owing to its cysteine protease activity towards a-tubulin [32]. However, the structure of VirA does not exhibit any similarity to papain-like cysteine proteases, and the role of purified VirA in the degradation of tubulin or disassembly of microtubules has not been confirmed [33,34].
Effectors produced in response to T3SA activation Effectors whose production is dependent upon MxiE and the T3SA activity include OspD3/SenA, OspE1, OspE2, OspG and members of the IpaH family. Inactivation of mxiE did not affect the ability of bacteria to enter epithelial cells, suggesting that these effectors are not required for entry [10]. Unexpectedly for a T3SA substrate, secreted OspD3/SenA, i.e. present in the culture medium, was shown to be endowed with an enterotoxin activity [39]. OspE proteins are encoded by two almost identical genes (ospE1 and ospE2) in S. flexneri. In S. sonnei, ospE1 carries a frameshift mutation and inactivation of ospE2 did not affect the ability of bacteria to enter epithelial cells but led to the formation of smaller plaques on a confluent cell monolayer. Cells infected for 3 h by the ospE2 mutant, but not the wild-type strain, exhibited an altered morphology characterized by a rounding shape. A HAtagged OspE2 protein produced by S. sonnei accumulated at focal contact-like structures in infected cells [40]. Current Opinion in Microbiology 2009, 12:110–116
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OspG exhibits sequence similarities with protein kinases and was shown to be endowed with an autophosphorylation activity and to interact with ubiquitinated E2 ubiquitin conjugating enzymes (Ubc), including UbcH5 and UbcH7. In transfected cells, production of OspG prevented degradation of IkBa induced by TNF-a, suggesting that OspG interferes with ubiquitination of p-IkBa by the E3 ubiquitin ligase SCFb-TrCP. As compared with the wild-type strain, an ospG mutant induced a faster degradation of p-IkBa in infected cells and a stronger inflammatory response in rabbit ligated ileal loops [41]. IpaH proteins are characterized by a variable N-terminal domain containing six to eight Leucine-rich repeats (LRR) and a conserved C-terminal domain of 300 residues. In yeast, production of IpaH9.8 interrupted the pheromone response pathway by inducing the proteasome-dependent degradation of the MAPK kinase Ste7 and, in vitro, IpaH9.8 was shown to be endowed with an E3 ubiquitin ligase activity towards Ste7 [42]. The catalytic activity of the IpaH C-terminal domain is dependent upon a Cys residue involved in the transfer of ubiquitin to the substrate through a transthiolation reaction, as described for HECT domain containing E3s; this domain does not exhibit any structural similarities with eukaryotic E3s [43,44]. Differences in the substrate specificity of IpaH9.8, IpaH1.4 and SspH1 (a Salmonella enterica IpaH homologue) suggested that the LRR-containing N-terminal domain corresponds to the substrate recognition domain [42,43]. Intriguingly, the C-terminal domain of IpaH9.8 was shown to interact with the splicing factor U2AF35 [45]. Following intranasal infection of mice, an ipaH9.8 mutant and a mutant in which all chromosomal ipaH genes were inactivated provoked a stronger inflammatory response and a reduced bacterial colonization than the wild-type strain [45,46].
bacteria do not enter, rather than a post-invasion stage, i.e. in infected cells. Remarkable progresses have been made in elucidating the structure and deciphering the function of a number of Shigella effectors. Once a catalytic activity, or a binding partner, has been identified, there might be a long way between the characterization of a substrate and the identification of the substrate whose modification has been selected throughout evolution as beneficial to the pathogen. Even though generalizations must be taken cautiously, trends seem to emerge; some effectors promote entry of bacteria within epithelial cells, whereas others dampen innate immune responses induced upon infection. Interestingly, effectors involved in entry are produced independently of the T3SA activity, whereas effectors involved in controlling cell signaling are produced in response to activation of the T3SA. Unraveling the functions of effectors points to the how and the for what purposes; we still have much to learn on the where and the when effectors are effective.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Penno C, Sansonetti P, Parsot C: Frameshifting by transcriptional slippage is involved in production of MxiE, the transcription activator regulated by the activity of the type III secretion apparatus in Shigella flexneri. Mol Microbiol 2005, 56:204-214.
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Penno C, Hachani A, Biskri L, Sansonetti P, Allaoui A, Parsot C: Transcriptional slippage controls production of type III secretion apparatus components in Shigella flexneri. Mol Microbiol 2006, 62:1460-1468.
8.
Buchrieser C, Glaser P, Rusniok C, Nedjari H, d’Hauteville H, Kunst F, Sansonetti P, Parsot C: The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri. Mol Microbiol 2000, 38:760-771.
Concluding remarks Control of effector-encoding genes by the T3SA activity imposes a hierarchy in the delivery of effectors to target cells; effectors produced before activation of the T3SA should transit before effectors produced in response to activation of the T3SA. However, except for effectors involved in entry, we do not know the stage of the infection at which effectors are most needed; is it when bacteria are within epithelial cells, or when they are facing inflammatory cells recruited to the site of infection, or at the onset of infection to promote bacterial colonization and survival? MxiE-controlled effectors delivered at the apical side of epithelial cells by presumably extracellular bacteria were shown to suppress transcription of genes encoding antimicrobial peptides and chemokines and, upon infection of human intestinal xenotransplants in mice, to promote progression of bacteria towards intestinal crypts [47]. Thus, MxiE-controlled effectors might be involved at a pre-invasion stage, i.e. in cells in which Current Opinion in Microbiology 2009, 12:110–116
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Enninga J, Mounier J, Sansonetti P, Van Nhieu GT: Secretion of type III effectors into host cells in real time. Nat Methods 2005, 2:959-965. This study presents a novel approach to monitor transit of T3SA substrates in real time and shows that, following contact of bacteria with epithelial cells, the T3SA is activated within seconds. www.sciencedirect.com
Shigella type III secretion effectors: how, where, when, for what purposes? Parsot 115
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of GTP-active GTPases of the Ras family, IpgB1, IpgB2 and Map functioning as active forms of RhoA, Rac1 and Cdc42, respectively (see also Reference [26]). 26. Handa Y, Suzuki M, Ohya K, Iwai H, Ishijima N, Koleske AJ, Fukui Y, Sasakawa C: Shigella IpgB1 promotes bacterial entry through the ELMO-Dock180 machinery. Nat Cell Biol 2007, 9:U121-U165. This study shows that IpgB1 associates with ELMO and Dock180 to stimulate Rac1 activity and promote membrane ruffling at the site of bacterial entry; IpgB1 is proposed to act as a RhoG mimic (see also Reference [25]). 27. Slagowski NL, Kramer RW, Morrison MF, LaBaer J, Lesser CF: A functional genomic yeast screen to identify pathogenic bacterial proteins. Plos Pathogens 2008:4. By monitoring growth of a collection of yeast expressing virulence plasmid-encoded proteins, the authors propose growth inhibition of yeast as a new assay to identify T3S effectors. 28. Zurawski DV, Mumy KL, Badea L, Prentice JA, Hartland EL, McCormick BA, Maurelli AT: The NleE/OspZ family of effector proteins is required for polymorphonuclear transepithelial migration, a characteristic shared by enteropathogenic Escherichia coli and Shigella flexneri infections. Infect Immun 2008, 76:369-379. 29. Santapaola D, Del Chierico F, Petrucca A, Uzzau S, Casalino M, Colonna B, Sessa R, Berlutti F, Nicoletti M: Apyrase, the product of the virulence plasmid-encoded phoN2 (apy) gene of Shigella flexneri, is necessary for proper unipolar IcsA localization and for efficient intercellular spread. J Bacteriol 2006, 188:1620-1627. 30. Zurawski DV, Mitsuhata C, Mumy KL, McCormick BA, Maurelli AT: OspF and OspC1 are Shigella flexneri type III secretion system effectors that are required for postinvasion aspects of virulence. Infect Immun 2006, 74:5964-5976. 31. Yoshida S, Katayama E, Kuwae A, Mimuro H, Suzuki T, Sasakawa C: Shigella deliver an effector protein to trigger host microtubule destabilization, which promotes Rac1 activity and efficient bacterial internalization. EMBO J 2002, 21:2923-2935. 32. Yoshida S, Handa Y, Suzuki T, Ogawa M, Suzuki M, Tamai A, Abe A, Katayama E, Sasakawa C: Microtubule-severing activity of Shigella is pivotal for intercellular spreading. Science 2006, 314:985-989. 33. Germane KL, Ohi R, Goldberg MB, Spiller BW: Structural and functional studies indicate that Shigella VirA is not a protease and does not directly destabilize microtubules. Biochemistry 2008, 47:10241-10243. 34. Davis J, Wang J, Tropea JE, Zhang D, Daute Z, Waugh DS: Wlodawer A: Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri. Protein Sci 2008, 17:2167-2173. 35. Li HT, Xu H, Zhou Y, Zhang J, Long CZ, Li SQ, Chen S, Zhou JM, Shao F: The phosphothreonine lyase activity of a bacterial type III effector family. Science 2007, 315:1000-1003. This study demonstrates the phosphothreonine lyase activity of OspF that irreversibly inactivates the dually phosphorylated MAPKs Erk1/2, p-38 and JNK in vitro (see also References [36,37] and the follow-up in Reference [38]). 36. Arbibe L, Kim DW, Batsche E, Pedron T, Mateescu B, Muchardt C, Parsot C, Sansonetti PJ: An injected bacterial effector targets chromatin access for transcription factor NF-kappa B to alter transcription of host genes involved in immune responses. Nat Immunol 2007, 8:47-56. 37. Kramer RW, Slagowski NL, Eze NA, Giddings KS, Morrison MF, Siggers KA, Starnbach MN, Lesser CF: Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation. Plos Pathogens 2007, 3:179-190. 38. Zhu YQ, Li HT, Long CZ, Hu LY, Xu H, Liu LP, Chen S, Wang DC, Shao F: Structural insights into the enzymatic mechanism of the pathogenic MAPK phosphothreonine lyase. Mol Cell 2007, 28:899-913. This structural study of wild-type SpvC and an inactive SpvC variant in complex with a phosphopeptide substrate, as well as a detailled enzyCurrent Opinion in Microbiology 2009, 12:110–116
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