Biochemistry and cell signaling taught by bacterial effectors

Review

Biochemistry and cell signaling taught by bacterial effectors Jixin Cui and Feng Shao National Institute of Biological Sciences, 7# Science Park Road, Zhongguancun Life Science Park, Beijing, 102206, China

Bacterial virulence often relies on secreted effectors that modulate eukaryotic signal transduction. Recent studies provide a collection of examples in which bacterial effectors carry out unprecedented posttranslational modifications of key signaling molecules or organize a new signaling network. OspF and YopJ families of effectors use novel modification activities to block kinase phosphoactivation. Targeting of the ubiquitin system by IpaH and Cif/CHBP families provides insights into host ubiquitin signaling. Manipulation of small GTPases by VopS/IbpA and SidM suggests previously underappreciated regulation of signaling. Several other effectors, including SifA and EspG, organize newly discovered signaling networks in membrane trafficking. Studies of these effectors can generate new knowledge in enzyme catalysis and provide new angles for furthering our understanding of biochemical regulation of important signaling pathways. Introduction to bacterial effectors and their modulation of host signal transduction systems Some Gram-negative bacterial pathogens harbor specialized secretion systems such as type III (TTSS) and type IV secretion systems to inject effector proteins into host cells [1,2]. These effectors, which arose from the long evolutionary fight between host and pathogen, employ sophisticated strategies to manipulate eukaryotic signaling pathways and thereby thwart host defenses. Revelation of the functional mechanisms of bacterial effectors not only leads to a better understanding of bacterial virulence strategies, but also has general significance for learning about new cellular enzyme activities and gaining further knowledge of eukaryotic signal transduction. Bacterial effectors have a tropism for several key signaling pathways in host cells. On sensing bacterial infection, the host innate immune system transcriptionally upregulates many inflammatory cytokines, usually through activation of the mitogen-activated protein kinase (MAPK) pathway or nuclear factor-kB (NF-kB) signaling, both of which involve multiple phosphorylation events mediated by different kinases. The large majority of host cellular processes including the immune defense are regulated by ubiquitylation, a posttranslational modification that often leads to protein destruction in the proteasome. Numerous studies now suggest that the MAPK and NF-kB pathways, as well as the ubiquitin system, are frequently targeted by bacterial effectors [3,4]. Moreover, it has long Corresponding author: Shao, F. ([email protected])

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been known that bacterial effectors modify the Rho family of small GTPases to manipulate host actin cytoskeleton dynamics. Recent studies have demonstrated that targeting of Rab and ARF GTPases signaling is crucial for the interplay between intracellular pathogens and host membrane trafficking. Here we discuss recent findings on bacterial effectors that carry out new posttranslational modifications of key signaling molecules or rewire signaling networks in host cellular processes regulated by MAPK, NF-kB, ubiquitin and small GTPases. New modes of counteracting phosphoactivation of kinases involved in MAPK and NF-kB signaling The MAPK cascade, consisting of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs) and MAPK, plays important roles in innate immune defense. MAPKs are generally divided into three groups: extracellular signalregulated protein kinase (ERK), p38 and c-Jun N-terminal kinase (JNK). MAPKs are activated by MAPKK-catalyzed dual phosphorylation of a T-X-Y motif (X is E in ERK, G in p38 and P in JNK) in the kinase activation loop. MAPK phosphatases (MKPs) remove phosphates from the pT-X-pY motif to negatively regulate signaling. The NF-kB pathway requires MAPKK-like IkB kinase (IKK) for activation of downstream signaling. Here we discuss two modes of regulating kinase activation in MAPK and NF-kB signaling used by the OspF and YopJ families of TTSS effectors. The OspF family of phosphoserine/phosphothreonine lyases: elimination of phosphothreonine in MAPKs and beyond The OspF family effectors, including OspF from Shigella flexneri, SpvC from Salmonella spp., VirA from Chromobacterium violaceum and HopAI1 from the plant pathogen Pseudomonas syringae, suppress host MAPK signaling, and in the case of Shigella infection, downregulate interleukin (IL)-8 expression [5–8]. The OspF family efficiently removes the threonine phosphate from the pT-X-pY motif in activated MAPKs. OspF catalyzes a b elimination reaction to convert the phosphothreonine into b-methyldehydroalanine (Figure 1), which differs from cellular MKPs that catalyze phosphate hydrolysis. The OspF family defines a novel phosphothreonine lyase [7] and the resulting modification is termed eliminylation [9]. OspF-like phosphothreonine lyase has a unique a/b-fold structure with no precedents [10]. Part of the N-terminal type III secretion signal in OspF, SpvC and VirA mimics the canonical D motif used by MAPK substrates, activators and regulators to recognize MAPKs. Structural analysis has

0968-0004/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2011.07.003 Trends in Biochemical Sciences, October 2011, Vol. 36, No. 10

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MAPK signaling upstream

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Figure 1. Prevention of phosphoactivation of kinases in MAPK signaling by the YopJ and OspF families of effectors. YopJ acetylates conserved Ser/Thr in the MAPKK activation loop, and thereby occupies the sites of phosphorylation by upstream MAPKKK. OspF is a phosphothreonine lyase that converts the phosphothreonine within the MAPK pT-X-pY motif to b-methyldehydroalanine, which resists phosphorylation by MAPKK. The red asterisk denotes b-methyldehydroalanine derived from OspF-reacted phosphothreonine. Ac, acetyl group.

revealed a drastic conformational twist of the pT-X-pY motif, which releases the phosphothreonine from a highly positively charged pocket in MAPK and allows for insertion of phosphothreonine into a similar pocket in the lyase [10]. Thus, OspF exhibits a differential catalytic efficiency for ERK1/2, JNK and p38, which correlates with the nature of the X residue in the T-X-Y motif of different MAPKs. Moreover, extensive structural contacts with the phosphotyrosine and the threonine phosphate in the T-X-Y motif also contribute to specific substrate recognition by the lyase. The OspF family of phosphothreonine lyases employs a general acid–base catalytic mechanism. In an unusual manner, a lysine side-chain amine with pKa decreased to 7.9 acts as a general base to abstract the aproton, whereas a histidine acts as a general acid to protonate the leaving oxygen [10]. Elimination of the threonine hydroxyl group by the OspF family irreversibly terminates MAPK signaling, which probably benefits the pathogen. The resulting Ca=Cb following elimination is a good Michael acceptor that is sensitive to nucleophilic attack, particularly under the complex cellular context. In support of this idea, cysteamine readily reacts with the ‘threonine’ Ca=Cb in OspFtreated ERK2 phosphopeptide [10]. Such chemical alteration of the Ca=Cb could potentially create an additional gain-of-function signal, which might explain the OspFinduced epigenetic phenomenon of selective blocking of MAPK-dependent gene transcription [5]. The OspF family is the first example of reversal of phosphoactivation of kinases through b elimination of phosphothreonine. Questions arise as to whether such an enzymatic modification exists elsewhere and whether eliminylation regulates other signaling, particularly as a counteraction of serine/threonine phosphorylation. Lantibiotic synthetases, a group of enzymes responsible for biosynthesis of lantibiotics in lactic bacteria, introduce (b-methyl)dehydroalanine into polycyclic peptide antibiotics by dehydration of a serine/threonine [11].

Interestingly, the serine/threonine is also phosphorylated before dehydration, which indicates a similar b elimination reaction. Indeed, the recently identified class III lantibiotic synthetase is homologous to OspF and employs an OspF-like catalytic mechanism [12]. Furthermore, elimination of phosphoserine/phosphothreonine by lantibiotic synthetase is followed by nucleophilic attack of the Ca=Cb by a cysteine thiol to generate a thioether ring present in mature peptide antibiotics. This supports the hypothesis that OspF-inactivated MAPKs could further react with cellular nucleophilic agents. Another possible means to achieve b elimination of phosphoserine/phosphothreonine is through pyridoxal-50 phosphate (PLP)-mediated covalent catalysis. For instance, the PLP-dependent enzyme threonine synthase (EC 4.2.3.1) from bacteria and plants uses a Schiff basemediated catalysis to convert O-phosphohomoserine into a b,g-unsaturated intermediate [13]. PLP-dependent enzymes are widely present in nature. Homologs of threonine synthase are also found in mammals that do not require such enzymes for amino acid biosynthesis [14]. Therefore, the possibility of using PLP-dependent enzymes to generate eliminylation modification in higher eukaryotes probably exists. The YopJ family of serine/threonine acetyltransferases: blocking of phosphorylation and activation of the MAPKK superfamily The TTSS effector YopJ from Yersinia spp. compromises host defenses by inhibiting activation of MAPK and NF-kB pathways [15]. Differing from OspF, YopJ targets and inactivates the MAPKK superfamily, including IKK, in the NF-kB pathway. TTSS effectors homologous to YopJ are widely present in bacterial pathogens and symbionts, including AvrA from Salmonella spp., VopA from Vibrio parahaemolyticus, and AopP from Aeromonas salmonicida. Fold recognition analysis identified a putative cysteine protease catalytic triad in the YopJ family 533

Review (His-Asp/Glu-Cys), classified into Clan CE of cysteine proteases. The catalytic triad residues are essential for YopJ inhibition of MAPK and NF-kB signaling. YopJ transfers the acetyl group from acetyl-CoA to a conserved serine/threonine in MAPKKs, which prevents phosphorylation of these residues and activation of MAPKKs by MAPKKKs (Figure 1) [16,17]. YopJ also acetylates a threonine in the IKK activation loop and thereby disrupts NF-kB signaling. Other YopJ-like effectors harbor similar activities, but exhibit higher selectivity towards different MAPKKs. AvrA acetylates MAPKK4/7 and specifically blocks JNK signaling [18,19]; VopA and AopP abolish MAPK and NF-kB signaling, respectively, presumably through selective acetylation of different MAPKKs [20,21]. It is well established that lysine acetylation, namely N-acetylation, regulates many signaling pathways such as epigenetic regulation of gene transcription. Acetylation can also occur on the hydroxyl oxygen of a serine/threonine, referred to as O-acetylation. For example, serine acetyltransferase (SAT) synthesizes O-acetyl-serine from acetylCoA and serine, the first reaction step in cysteine synthesis in bacteria and plants [22]. YopJ also catalyzes O-acetylation, but on a protein side-chain hydroxyl group. YopJ is catalytically different from SAT and employs a cysteine nucleophile in the His-Asp/Glu-Cys triad to attack acetylCoA and form a thioester intermediate. This type of ping pong mechanism is used by other cysteine protease-like acetyltransferases, which usually catalyze N- rather than O-acetylation. Indeed, YopJ and VopA acetylate a lysine in MAPKK6 [17,21]; a YopJ-like effector from Ralstonia autoacetylates a lysine residue conserved in the YopJ family [23]. Therefore, the preference for O-acetylation probably results from substrate binding-induced appropriate positioning of a serine/threonine side chain into the catalytic center of the YopJ family. The catalytic and chemical similarity between N- and O-acetylation suggests that some eukaryotic acetyltransferases, particularly those that use a ping-pong mechanism, might also catalyze Oacetylation to regulate functions of the substrates. It is thought that YopJ is maintained in a quiescent state to avoid potential deleterious effects to the bacteria [24]. A recent report showed that YopJ and AvrA are allosterically activated by inositol hexakisphosphate [25], a eukaryotespecific small molecule. This raises the interesting hypothesis that other cysteine protease-like hydrolases might have similar allosteric activation. The YopJ family represents a new paradigm in signaling: O-acetylation of the kinase activation loop prevents the kinase from being phosphorylated and activated. Cysteine protease-like hydrolytic activities are abundant and ubiquitous, which suggests such a mechanism could possibly be used for eukaryotic signaling regulation. Moreover, VopA acetylates an additional lysine involved in coordinating the g-phosphate in MAPKK6 [21], which suggests inactivation of a kinase with an already phosphorylated activation loop and represents another mode of inhibition of kinase signaling. A genetic screen aimed at isolating YopJ-resistant variants of MAPKK identified a tyrosine!histidine mutant with elevated basal activity [26]. This mutant bypasses the requirement of phosphorylation 534

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for activation and will enrich our mechanistic understanding of MAPKK activation. Thus, the OspF family of phosphothreonine lyases and YopJ-like acetyltransferases induce eliminylation and O-acetylation of serine/threonine residues in host kinases, respectively, and block kinase phosphoactivation, which represent new models of regulation of kinase signaling. Bacterial insights into ubiquitin and ubiquitin-like systems Ubiquitylation regulates the majority of cellular processes including innate immune defense and is often a target of bacterial effectors. Ubiquitylation involves a three-enzyme cascade: the ubiquitin-activating enzyme E1 activates ubiquitin by forming a thioester bond between its catalytic cysteine and the C terminus of ubiquitin in an ATP-dependent manner. Ubiquitin is then transferred to the activesite cysteine in an E2 ubiquitin-conjugating enzyme through transthiolation. In the third step, an E3 ubiquitin ligase bridges the substrate and the ubiquitin-charged E2, and catalyzes the formation of an isopeptide bond between the ubiquitin C terminus and a lysine in the substrate or another ubiquitin. The nature of the ubiquitin chain linkage confers different fates to the substrate. The classical K48-linked chain targets the substrate for proteasomal degradation, whereas K63 or linear ubiquitin chains serve as a regulatory signal. Ubiquitin ligases are generally classified into HECT-domain and RING-domain families, with the majority belonging to the latter. The HECTdomain E3 uniquely requires a catalytic cysteine that forms a ubiquitin thioester intermediate. The RING-domain E3 exists as a single molecule or a multi-subunit form, with the latter represented by cullin-RING ubiquitin ligases (CRLs). The six mammalian cullins are all modified by a ubiquitin-like protein (UBL), NEDD8, on a C-terminal conserved lysine. NEDD8 monoconjugation (neddylation) stimulates CRL ubiquitin ligase activity [27]. Emerging evidence suggests that bacterial pathogens have evolved to exploit the ubiquitin system, for instance, by mimicking deubiquitin enzymes or ubiquitin ligases. Here we focus on IpaH and Cif/CHBP families of TTSS effectors because their mechanisms of action have shed new light on ubiquitin biochemistry and signaling. The IpaH family of bacterial ubiquitin ligases: a new strategy for synthesizing ubiquitin chains S. flexneri has a group of highly homologous TTSS effectors called IpaHs including IpaH3 and IpaH9.8 [28]. IpaHs belong to a large family of proteins from diverse bacterial pathogens including Salmonella, Yersinia and Pseudomonas, as well as Rhizobium-like symbionts. All IpaH family members consist of N-terminal leucine-rich repeats (LRRs) and a homologous C-terminal domain with potent ubiquitin ligase activity [29]. The IpaH ubiquitin ligase domain has an all-helical structure distinct from HECT and RING domains, and represents a third class of ubiquitin ligases [30–32]. Similar to HECT-domain E3 ligases, IpaH employs a catalytic cysteine within a conserved CXD motif, which forms an ubiquitin thioester during ubiquitin transfer from E2 to the substrate. Substitution of asparagine for aspartate

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IKKg) for ubiquitylation and degradation, which contributes to Shigella suppression of the NF-kB pathway [36]. IpaH9.8-synthesized ubiquitin chains on NEMO are linked predominantly via K27 (Figure 2), in contrast to predominantly K48-linked polyubiquitin chains in vitro [30,31,33]. Formation of K27 ubiquitin chains and their function in ubiquitin signaling and pathogen–host interactions have not yet been characterized. Thus, the unique properties of IpaH-like ubiquitin ligase effectors expand our knowledge about ubiquitin transfer mechanisms and might also reveal new biological implications.

in the CXD motif also abolishes the E3 activity of IpaH3 [31]. Notably, the mutant protein instead becomes a potent ubiquitin–E2 thioesterase in which the cysteine is the catalytic nucleophile. The negatively charged aspartate possibly helps to create a deprotonation-prone microenvironment at the catalytic center, which facilitates nucleophilic attack of the IpaH–ubiquitin thioester by the substrate lysine. A requirement of IpaH-like catalytic aspartate has not been observed for any eukaryotic ubiquitin ligase. This finding also suggests that IpaH-like bacterial E3 s might have evolved from a thioesterase ancestor given the wide presence of thioesterases in prokaryotes. IpaH prefers to use UbcH5 as the E2 in vitro [30,31], but recognizes a UbcH5 surface different from that used by eukaryotic E3 s [30,33]. SspH2, the Salmonella homolog of IpaH, builds a polyubiquitin chain tethered to the active-site cysteine in UbcH5, which results in extremely efficient substrate ubiquitylation kinetics [33]. LRR domains in the structures of IpaH3 and SspH2 show drastically different orientations relative to the ubiquitin ligase domain [31,32]. In SspH2, the LRR domain interacts with the ubiquitin ligase domain, which buries the catalytic cysteine. Consistently, full-length IpaH9.8 and SspH2 are less active than the ligase domain alone [30,32]. The LRR domain probably binds the substrate, which also releases its inhibitory effect on the ubiquitin ligase domain. The heterogeneity of LRR domains among IpaH family members predicts a diversity of substrates. It has been reported that the human splicing factor U2AF35 [34] and the kinase PKN1 [29,35] are in vitro substrates of IpaH9.8 and SspH2, respectively. IpaH9.8 also targets NEMO (also known as

The CHBP/Cif family of ubiquitin and NEDD8 deamidases: modulation of ubiquitin signaling by deamidation The Cif TTSS effector from enteropathogenic Escherichia coli (EPEC) and the homologous CHBP from Burkholderia pseudomallei induce a cytopathic effect on epithelial cells [37–39]. This cytopathic effect is characterized by cell cycle arrest and drastic morphological changes including cell enlargement and actin stress fiber formation. Structurally, Cif and CHBP adopt a papain-like cysteine protease fold with a Cys-His-Gln catalytic triad that is essential for the cytopathic effect [37–40]. CHBP efficiently inhibits polyubiquitin chain synthesis independent of E2 and E3 [41]. CHBP acts as a deamidase to convert Gln40 in ubiquitin to glutamate (Figure 2) [41]. Gln40-deamidated ubiquitin is largely intact during E1 and E2 charging, but severely impairs E3-catalyzed chain formation. This is the first appreciation of the functional importance of Gln40 in ubiquitin, which opens a new window for studying the

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Figure 2. Manipulation of the ubiquitylation system by the IpaH and CHBP/Cif families of effectors. Effector proteins are secreted from bacteria into the host cytoplasm through a syringe-like TTSS. The IpaH family of effectors defines a new class of ubiquitin ligases that assemble polyubiquitin chains. The CHBP/Cif family functions as glutamine deamidase to hydrolyze Gln40 in ubiquitin/NEDD8 to Glu. Deamidated ubiquitin is attenuated in supporting ubiquitin chain formation, whereas deamidated NEDD8 inhibits the activity of neddylated cullin-RING ubiquitin ligases (CRLs). Abbreviations: Ub, ubiquitin; N8, NEDD8; SRM, the substrate recognition module of CRL; ABIN, A20 binding inhibitor of NF-kB; Roc1, RING of cullin 1.

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ubiquitin transfer mechanism. Deamidated ubiquitin–E2 thioester could potentially allow for capture of the transition state of ubiquitin transfer during chain formation. NEDD8 is the only UBL that shares sequence homology with ubiquitin. CHBP also efficiently deamidates Gln40 in NEDD8 (Figure 2). Cif much prefers to deamidate NEDD8 [41], consistent with identification of NEDD8, but not ubiquitin, as a direct target of Cif [42,43]. Cif and CHBP do not target other UBLs for deamidation. Deamidation of NEDD8 by Cif and CHBP impairs degradation of multiple CRL substrates in vivo. NEDD8 deamidation is responsible for Cif- and CHBP-induced cell cycle arrest; ectopic expression of NEDD8 Q40E partially recapitulates the phenotype. These observations reemphasize the crucial role of CRLs, particularly the SCF (Skp1–cullin 1–F-box) complex, in regulating cell cycle progression. How neddylation stimulates CRL E3 ligase activity is not well understood, but probably results from neddylation-induced conformational rearrangement of the CRL complex [44]. Conjugation of cullin by deamidated NEDD8 completely abolishes rather than promotes the E3 ligase activity of CRLs. Thus, the bacterium has posed a stimulating question about the function of Gln40 in NEDD8. Answering this question, possibly by determining the NEDD8 Q40E-conjugated CRL complex structure, will generate insights into neddylation stimulation of CRL activation. Cif- and CHBPinduced stress fiber formation also results from NEDD8 deamidation, which suggests a crucial role for CRL in regulating actin cytoskeleton dynamics. This facilitated the discovery of a novel cullin 3–BACURD CRL complex that controls ubiquitylation and degradation of the small GTPase RhoA [45]. Thus, the ubiquitin or NEDD8 deamidase activity of Cif and CHBP provides a unique tool for probing the role of ubiquitylation or neddylation in other cellular processes. Regulation of small GTPase activity and signaling by stable adenylylation (AMPylation) The Ras superfamily of small GTPases, including the Rho, Rab and ARF families, regulates diverse pathways in

eukaryotes. Ras-like GTPases cycle between GTP-bound ‘on’ states and GDP-bound ‘off’ states, catalyzed by guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Nucleotide binding determines the conformational state of two flexible regions (switches I and II) in the GTPase. The switch regions are responsible for binding and signaling downstream effector activation. The Rho family, including RhoA, Rac and CDC42, controls actin cytoskeleton dynamics, whereas Rab and ARF families are crucial for membrane trafficking [46]. Bacterial manipulation of small GTPase function is crucial for many aspects of pathogenesis such as invasion and establishment of bacteria-containing vacuoles. Historically, bacterial toxins, particularly Clostridium botulinum exoenzyme C3, were instrumental for identification and functional establishment of small GTPases. Many bacterial effectors that directly modify small GTPases have now been identified [47]. Here we briefly discuss two new classes of bacterial proteins that induce a modification known as adenylylation or AMPylation on Ras-like small GTPases [48,49] in light of eukaryotic signal transduction. VopS and IbpA: Fic-domain adenylyl transferases VopS, a TTSS effector from the marine pathogen Vibrio parahaemolyticus, disrupts the host actin cytoskeleton by inactivating Rho GTPases [50]. VopS modifies Rho by adding an adenylyl group specifically onto the hydroxyl group of a conserved threonine (Thr37 in RhoA) (Figure 3) [51]. VopS-catalyzed modification is also termed AMPylation [51] to distinguish it from transient adenylylation. VopS harbors a C-terminal Fic (filamentation induced by cAMP) domain responsible for the catalytic activity. The conserved histidine in the Fic domain signature motif [HPFX(D/E)GN(G/K)R] is essential for VopS function and adenylyl transferase activity. Parallel to the discovery of VopS, it was demonstrated that Fic domains from a large secreted antigen IbpA from the respiratory pathogen Histophilus somni possess adenylyl transferase activity towards Rho GTPases [52]. IbpA contains two Fic domains in tandem and both of them seem to be active. Similar to

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Figure 3. Modulation of small GTPase activity and signaling by stable adenylylation catalyzed by two enzymatically different classes of bacterial proteins. The conserved Fic domain in Vibrio VopS and Histophilus IbpA proteins transfer the AMP moiety from ATP to conserved Thr/Tyr in the Rho GTPase switch I region, which blocks downstream effector recognition by the GTPase. The N-terminal domain of the Legionella type IV effector SidM (SidM NTD) is a structural mimic of E. coli glutamine synthetase adenylyl transferase (GS-ATase). SidM NTD adenylylates Rab1-GTP on a switch II tyrosine, which prevents the GTPase from GAP-catalyzed GTP hydrolysis and prolongs the duration of Rab1-GTP for activation of the Legionella LidA effector. The chemical diagram shows adenylylation modification of Ser/Thr/Tyr residues.

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Review VopS, the IbpA Fic domains prefer to modify Rho-GTP and the enzyme activity requires an equivalent histidine. IbpA Fic differs from VopS in that it selectively adenylylates a tyrosine (Tyr34 in RhoA). Both Thr37 and Tyr34 in RhoA are conserved within the switch I region, and the bulky AMP modification blocks Rho GTPase recognition of downstream effectors. Crystal structures of VopS and IbpA Fic2 show a similar helical Fic-domain fold despite the limited sequence homology outside of the HPFX(D/E)GN(G/K)R motif [53,54]. The IbpA Fic2–CDC42 complex structure reveals that CDC42 switch regions I and II provide the major interaction surface for IbpA Fic2 [54]. Hydrophobic contact of switch II observed for IbpA Fic2 probably applies to VopS, because a triple mutation in the CDC42 switch II region similarly affects adenylylation by both IbpA Fic2 and VopS. Detailed structural comparison predicts that VopS and IbpA Fic2 adopt different switch I binding modes, which could explain why the two enzymes adenylylate different switch I residues. Despite an active-site histidine mutant used for crystallization, the IbpA Fic2 structure indeed captures an end product of an adenylylation reaction. This supports the proposed enzymatic mechanism for Fic domain-catalyzed adenylylation, in which the substrate tyrosine nucleophilically attacks the ATP a-phosphate and the histidine in the HPFX(D/E)GN(G/K)R motif acts as a general base to deprotonate the tyrosine hydroxyl group [54]. The hypothesis is strengthened by kinetic analysis of the initial velocity for VopS-catalyzed CDC42 adenylylation [53]. Moreover, a recently reported structure of another Fic-domain adenylyl transferase, BepA from Bartonella henselae, identified the substrate binding surface for the b- and g-phosphates of ATP [55]. The VopS-modified threonine is also targeted by Clostridium difficile toxin B, which this inactivates Rho GTPases by glycosylation [47]; this highlights the importance of this threonine in Rho signaling. The IbpA Ficmodified tyrosine in CDC42 undergoes a drastic and unprecedented conformational change to allow for adenylyl transfer [54]. This stresses the extreme conformational plasticity of switch I in Rho GTPases. There are over 2700 Fic-domain proteins spread throughout all kingdoms of life, with the majority from prokaryotes [54], which suggests wide use of adenylylation modification in bacteria. SidM/DrrA: combination of GS-ATase-like adenylyl transferase and GEF/GDF for Rab GTPases SidM (also known as DrrA) is a type IV effector from intracellular Legionella pneumophila, which infects alveolar macrophages and cause Legionnaires’ disease [56]. SidM was identified from searches for Legionella effectors that recruit Rab1 and interact with host endoplasmic reticulum-derived vesicles [57,58]. SidM was first shown to be capable of catalyzing the exchange of GDP for GTP and GDI (GDP-dissociation inhibitor) displacement from Rab1 [59,60]. Binding of Rab1 by a novel Rab1-activation domain in SidM induces drastic displacement of switch I, which triggers coupled GDP release and GDI displacement [61–63]. The N-terminal domain (NTD) preceding the Rab1activation domain in SidM is toxic to eukaryotic cells

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[58]. A crystal structure of SidM NTD revealed unexpected similarity to the catalytic domain of glutamine synthetase adenylyl transferase (GS-ATase) [64,65]. The GS-ATase catalytic motif G-X11-D-X-D is also present in the SidM NTD. The SidM NTD is indeed capable of transferring AMP to the conserved Tyr77 in the switch II region of Rab1-GTP (Figure 3) [64]. The modification does not affect the active conformation state of Rab1, but inhibits GAPstimulated GTP hydrolysis. Thus, SidM secures Rab1 in the active state following nucleotide exchange-mediated activation by its Rab1-activation domain, which highlights the finely tuned bacterial control of eukaryotic signaling. SidM NTD and the Fic domain represent two examples of stable adenylylation, and add another layer to regulation of small GTPase signaling. Notably, a separate adenylyl removase domain in GS-ATase catalyzes GS deadenylylation through phosphorolysis [66]; Ibp Fic-modified Rho can be deadenylylated by a phosphodiesterase via hydrolysis [52]. These observations suggest a potential reversible nature of adenylylation. An important question is whether adenylylation regulates signaling in eukaryotes. Humans have one Fic-domain protein called HYPE (huntingtin yeast-interacting protein E) that is ubiquitously expressed and catalytically active [52]. SidM NTD-like structural mimicry of nucleotidyl transferases that often escape from sequence homology-based identification might also exist in eukaryotes. Interestingly, overexpression of HYPE has no effect on the actin cytoskeleton, which suggests that endogenous stable adenylylation in humans regulates signaling other than Rho GTPases. Future identification of more adenylylation enzymes and proteomic efforts using adenylylation-specific antibodies [67] will aid in establishing the role of stable adenylylation in signaling. Newly identified signaling networks organized or routed by bacterial effectors In addition to manipulating eukaryotic signaling by covalently modifying key signaling molecules, bacterial effectors also rewire host signaling by serving as a new signaling node or a catalytic scaffold. In a survey of bacterial effectors that regulate vesicle trafficking, EspG from enterohemorrhagic E. coli (EHEC) was identified as a cisGolgi-localized exocytosis inhibitor that triggers severe Golgi fragmentation [68]. EspG targets ARF-GTP through binding to the switch I region and nucleotide-binding pocket. EspG binding prevents ARF-GAP-catalyzed GTP hydrolysis (Figure 4). EspG also targets p21-activated kinases (PAKs) using a different surface from ARF interactions. EspG recognizes a PAK autoinhibitory region that is normally targeted by host Rac1/CDC42 to induce PAK activation [68,69]. PAKs are not known to function in Golgi trafficking and PAK2 is directed to the Golgi structure due to the formation of a ternary ARF–EspG–PAK complex. Whereas the functional consequence of PAK activation at the Golgi surface remains undefined, the proposed catalytic scaffold action of EspG creates a new GTPase–kinase complex (Figure 4) [68]. Somewhat similarly to EspG, EspF (another EPEC/EHEC TTSS effector) simultaneously interacts with neuronal Wiskott-Aldrich syndrome protein (N-WASP) and the SH3 domain of sorting nexin 9 (SNX9) using its different proline-rich repeats [70]. By nucleating a 537

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Figure 4. Assembly of new signaling complexes and signaling rewiring by bacterial effectors for regulation of membrane dynamics. (a) EspG from EHEC directly targets GTP-bound ARF GTPases, which results in inhibition of ARF-GAP function and fragmentation of the Golgi apparatus. Concurrently, EspG also recruits and activates p21activated kinase (PAK), which generates an ARF–EspG–PAK ternary complex on the Golgi membrane. (b) Salmonella effectors SifA and SseJ locate on the cytoplasmic face of a Salmonella-containing vacuole (SCV) and regulate SCV dynamics. The N terminus of SifA binds to SifA kinesin interacting protein (SKIP), which connects the SCV to host microtubules (MTs) and plays an important role in SCV dynamics. Meanwhile, the C-terminal putative GEF domain in SifA specifically recognizes RhoA-GDP and probably converts it to RhoA-GTP. The activated RhoA in turn interacts with SseJ and stimulates the phospholipase and glycerophospholipid cholesterol acyltransferase (GCAT) activity of SseJ. The enzyme activity of SseJ then regulates the lipid composition of the SCV membrane. SseJ hydrolyzes and transfers the fatty acid in the sn-1 position (red) of a phospholipid, as illustrated in the chemical diagram.

SNX9–EspF–N-WASP ternary complex, EspF coordinates SNX9-mediated membrane remodeling with N-WASP-controlled filamentous actin assembly. Assembly of a multimeric effector signaling complex can also be mediated by a host scaffold protein. In EHEC, infection-induced pedestal actin requires translocated intimin receptor (Tir) and EspF-like effector EspFU, which are physically connected by IRTKS (insulin receptor tyrosine kinase substrate) or the closely related IRSp53 [71,72]. EspFU also recruits and activates N-WASP. Formation of the Tir–IRTKS/IRSp53– EspFU complex beneath the bacterial attachment site regulates N-WASP-dependent and -independent actin polymerization, and possibly other signaling events [73]. Bacterial effectors also rewire eukaryotic signaling to catalyze lipid modification and regulate membrane dynamics. Intracellular Salmonella replicates within a specialized Salmonella-containing vacuole (SCV). SCV maturation and stability require Salmonella-induced tubular networks that extend from the SCV. Salmonella-induced filaments are crucial for the tubular structure, and the functions of two TTSS effectors, SifA [74] and SseJ [75,76], are involved in this. The C terminus of SifA is modified by host prenylation and S-acylation machinery, which thereby localizes SifA to the SCV cytoplasmic face [77,78]. The SifA N-terminal domain recruits a kinesin-interacting protein (SKIP) [78– 80]. This connects SCVs to microtubules and promotes vesicle budding from SCVs (Figure 4) [81]. The SifA Cterminal domain structurally mimics GEF for Rho GTPases [79,80]. SseJ is a GDSL-motif-containing lipase and exhibits deacylase, phospholipase and glycerophospholipid cholesterol acyltransferase (GCAT) activities in vitro [82–84]. SseJ specifically interacts with RhoA-GTP, which could 538

be generated by the presumed GEF activity of SifA [79,85]. RhoA binding stimulates the lipase and GCAT activities of SseJ [85], and thereby alters the cholesterol composition of the SCV membrane. Coexpression of SseJ and SifA in eukaryotic cells produces SKIP-dependent endosomal tubules [79], which partially recapitulates SCV tubulation. Therefore, SifA and SseJ engage host SKIP and RhoA in a highly sophisticated manner, which regulates SCV membrane dynamics and tubulation (Figure 4). Taken together, formation of the ARF–EspG–PAK signaling complex at the Golgi surface and generation of the SKIP–SifA–RhoA!SseJ signaling axis in membrane trafficking highlight an emerging new concept that bacterial effectors can serve as a new node to rewire host signal transduction and organize a new network. Concluding remarks and future perspectives We have discussed several stimulating examples of bacterial effectors that employ diverse and unique biochemical strategies to modulate eukaryotic signal transduction. These findings enrich our understanding of key signaling events such as phosphorylation, ubiquitylation and small GTPase switches, as well as related MAPK, NF-kB, ubiquitin and membrane trafficking signaling. The mechanism of action of bacterial effectors is generally thought to mimic at least some aspects of host function. If this is true, these effectors will certainly guide future efforts to elucidate eukaryotic signaling regulation. Given the diverse spectrum of bacterial pathogens and the large bacterial virulence arsenal, bacterial effector studies will continue to uncover new enzyme activities and posttranslational modifications and reveal new mechanisms in cell signaling.

Review Acknowledgements We apologize to colleagues whose work could not be cited owing to space limitations and the defined focus of the review. Work in the our laboratory is supported by National Basic Research Plan of China 973 grants.

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