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FEMS Microbiology Letters 188 (2000) 1^6 www.fems-microbiology.org MiniReview Bacterial protein toxins targeting Rho GTPases Maria Lerm, Gudula Schmidt, Klaus Aktories * Institut fu«r Pharmakologie und Toxikologie der Universita«t Freiburg, Hermann-Herder-Str. 5, D-79104 Freiburg, Germany Received 21 March 2000; received in revised form 2 May 2000; accepted 2 May 2000 Abstract Several bacterial protein toxins target eukaryotic cells by modulating the functions of Rho GTPases that are involved in various signal processes and in the regulation of the actin cytoskeleton. The toxins inhibit Rho functions by ADP-ribosylation or glucosylation and activate them by deamidation and transglutamination. New findings indicate that the GTPases are also targeted by various `injected' toxins which are introduced into the eukaryotic cells by the type-III secretion system. The injected toxins do not covalently modify Rho GTPases, but manipulate their regulatory GTPase cycle by acting as GTPase-activating proteins or guanine nucleotide exchange factors. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Rho GTPase ; Toxin; Glucosylation ; Type-III secretion ; ADP-ribosylation; Clostridium di¤cile toxin; Deamidation; Transglutamination ; Cytotoxic necrotising factor 1. Introduction Rho proteins comprise a family of low molecular mass GTPases (also called Ras superfamily of small GTPases) which act as molecular switches in a large array of signalling processes (for review see [1^3]). More than 10 Rho family members have been identi¢ed, which are at least 40% identical in their amino acid sequence. Best characterised are the Rho, Rac and Cdc42 subtypes and their respective isoforms. Like all Ras-related proteins, the Rho GTPases cycle between an inactive, GDP-bound state and an activated GTP-bound state. In the GDP-bound form, Rho GTPases are localised in the cytoplasm complexed with the guanine nucleotide dissociation inhibitor (GDI). An extracellular stimulus (e.g. lysophosphatidic acid) triggers the translocation of Rho from the cytosol to the plasma membrane. There, the Rho proteins dissociate from GDI and GDP/GTP exchange is catalysed by guanine nucleotide exchange factors (GEFs) resulting in the active GTP-bound form. The activated state of Rho proteins is terminated by the action of GTPase-activating proteins (GAPs), which stimulate the GTP hydrolysing activity of Rho. The structures of several small GTPases have been elucidated [4]. All members of the Ras superfamily possess two loops, the switch-I region covering ami- * Corresponding author. Tel. : +49 (761) 203 53 01; Fax: +49 (761) 203 53 11; E-mail : [email protected] no acids 28^38 of Rho and the switch-II region (covering amino acids 61^78) which both undergo considerable conformational changes depending on the nucleotide (GDP/ GTP) bound. In their active GTP-bound form, Rho GTPases interact with numerous e¡ectors, which include serine/threonine kinases, lipid kinases, phospholipase D and several adaptor proteins. Among the various described functions of Rho GTPases (e.g. control of secretion, endocytosis, transcriptional activation, cell cycle progression, transformation and apoptosis), the involvement of Rho GTPases in the regulation of the actin cytoskeleton is best studied [5]. RhoA induces formation of stress ¢bres and focal adhesions in mammalian ¢broblasts, whereas Cdc42 induces formation of microspikes and Rac leads to formation of lamellipodia and membrane ru¥es. Rho proteins are the targets of various bacterial proteins toxins (Table 1). Clostridium botulinum C3 ADP-ribosyltransferase, the related C3-like exoenzymes and the glucosyltransferases of the family of large clostridial cytotoxins (LCCs), including Clostridium di¤cile toxin A and B, inactivate Rho GTPases by covalent modi¢cation thereby causing major changes in the structure of the actin cytoskeleton and impairment of GTPase-controlled signalling. Rho GTPases are activated by deamidation or transglutamination catalysed by the cytotoxic necrotising factors (CNF) 1 and 2 from Escherichia coli and the dermonecrotic toxin (DNT) from Bordetella bronchiseptica and Bordetella parapertussis. Recently, several type-III sys- 0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 2 0 1 - 9 FEMSLE 9431 14-6-00 Cyaan Magenta Geel Zwart 2 M. Lerm et al. / FEMS Microbiology Letters 188 (2000) 1^6 Table 1 Toxins targeting Rho GTPases the release mechanism from GDI. Therefore, its ability to interact with the membrane receptors is impaired. Translocation of Rho to the plasma membrane and thus the activation of Rho will not occur [14]. Although C3 is a widely used tool to study signal pathways in which Rho is involved, use of C3 is hampered because it lacks a cellbinding and translocation domain (Figs. 1 and 2). The Toxins inactivating Rho GTPases by covalent modi¢cation C3-like ADP-ribosyltransferases : C. botulinum C3 exoenzyme C. limosum transferase B. cereus transferase S. aureus transferase Large clostridial cytotoxins: C. di¤cile toxin A C. di¤cile toxin B C. sordellii haemorrhagic toxin C. sordellii lethal toxin C. novyi K-toxin Toxins activating Rho GTPases by covalent modi¢cation Deamidating toxins: E. coli CNF1 (CNF2) E. coli CNF2 (CNF1) Transglutaminating/deamidating toxin: B. bronchiseptica, B. parapertussis, B. pertussis DNT `Injected' toxins non-covalently acting on Rho GTPases GAP-like `injected' toxins: P. aeruginosa exoenzyme S (ExoS) S. typhimurium protein tyrosine phosphatase (SptP) Y. pseudotuberculosis outer protein E (YopE) GEF-like `injected' toxin: S. typhimurium Sop E tem-injected bacterial toxins have been described to act on Rho proteins, either inhibiting (ExoS, YopE, SptP) or activating (SopE) the GTPases (Table 1). 2. Rho-inactivating toxins 2.1. Rho-ADP-ribosylating toxins The C3 exoenzyme from C. botulinum was the ¢rst bacterial `toxin' to be identi¢ed to block the biological functions of Rho GTPases [6^8]. Meanwhile several C3-related exoenzymes have been described produced by Clostridium limosum, Bacillus cereus and Staphylococcus aureus [9]. All these C3 exoenzymes are basic V25 kDa proteins that ADP-ribosylate RhoA, B and C but not other Rho family GTPases (e.g. Cdc42 and Rac) [10]. C3 exoenzymes ADPribosylate Rho at Asn41 [11] thereby blocking Rho-mediated signalling. Although the target amino acid residue is located near the e¡ector region of Rho, ADP-ribosylation does not block the Rho/e¡ector interaction [12] nor are major e¡ects on guanine nucleotide binding and GTP hydrolysis observed. Recent data indicate that it is rather the activation of Rho by its GEF (e.g. Lbc) that is impaired by this modi¢cation [12]. It is generally accepted that Rho requires translocation to the plasma membrane to become activated. The ezrin, radixin and moesin proteins are suggested to act as membrane `receptors' for Rho, releasing Rho from the GDI complex and exposing it to its GEFs [13]. In C3-treated cells, ADP-ribosylated Rho accumulates in the cytoplasm because ADP-ribosylation disturbs FEMSLE 9431 14-6-00 Fig. 1. Rho-targeting toxins. Bacterial protein toxins a¡ect Rho GTPases by covalent modi¢cation (1, 2) and by non-covalent modulation (3). Rho GTPases are inhibited by glucosylation catalysed by the family of LCCs. These toxins appear to contain at least three functional regions. Region I harbours the glucosyltransferase activity, region II and III might be responsible for transmembrane transport and receptor binding, respectively. The C3-like ADP-ribosyltransferases consist only of the catalytic domain. The Rho GTPase-activating toxins CNF and DNT contain three domains. The enzyme domain is located at the C-terminus (domain I). Domain III and II are suggested to be involved in receptor binding and translocation, respectively. CNF is a deamidase and DNT acts preferentially as a transglutaminase. The `injected' toxins are delivered into the target cells by the type-III secretion system. They possess a N-terminal translocation region for type-III secretion. The Rho inhibiting toxins (ExoS, SptP and YopE) possess a GAP-like domain whereas the Rho-activating toxin SopE harbours a GEF region. ExoS and SptP are multifunctional proteins and possess an ADP-ribosyltransferase domain and a tyrosine phosphatase domain, respectively. Cyaan Magenta Geel Zwart M. Lerm et al. / FEMS Microbiology Letters 188 (2000) 1^6 Fig. 2. Molecular mechanism of Rho-targeting toxins. Rho GTPases are inactive in the GDP-bound form. This form is stabilised by GDI. Activation of Rho occurs by GEF-stimulated release of GDP and binding of GTP. In the GTP-bound form Rho GTPases interact with multiple e¡ectors. The active state of Rho GTPases is terminated by hydrolysis of bound GTP. This is facilitated by GAPs. C3 ADP-ribosyltransferase modi¢es Rho (but not Rac and Cdc42) at Asn41 thereby inhibiting the activation by GEF proteins, and increases the a¤nity of Rho for GDI, which may also impair activation. ADP-ribosylated Rho still binds to e¡ectors, although, e¡ector activation might be disturbed. Glucosylation of Rho GTPases by LCCs at Thr37/Thr35 mainly disrupt the interaction of Rho GTPases with e¡ectors. In addition, in vitro experiments show that glucosylation blocks GEF-mediated activation of Rho and intrinsic and GAP-stimulated GTP hydrolysis. E. coli CNF 1 and 2 deamidates Rho GTPases at Gln63/Gln61. This inhibits the intrinsic and GAP-stimulated GTP hydrolysing activity of Rho proteins, making the GTPases constitutively activated. Bordetella DNT causes transglutamination of Rho GTPases at the same amino acid (Gln63/Gln61) of Rho GTPases. Like CNF, the functional consequence of DNT action is a constitutive activation of Rho proteins. S. typhimurium SptP, P. aeruginosa ExoS and Yersinia pseudotuberculosis YopE are GAPs which facilitate GTP hydrolysis by Rho proteins thereby inactivating the proteins. Finally, Salmonella SopE acts as a Rho GEF protein to facilitate the release of Rho-bound GDP and causes the binding of GTP to activate Rho. toxin must be applied at rather high concentrations to intoxicate cultured cells. Therefore, most studies performed with C3 use microinjection. Recently, a transport system for C3 was developed by means of the C. botulinum C2 toxin. This toxin is a two-component toxin consisting of two separate polypeptide chains, one of which harbours the actin-speci¢c ADP-ribosyltransferase activity (C2I) and the other, the cell-binding and toxin up-take functions (C2II) [15]. C3 exoenzyme was fused to the C2II-binding part of C2I, thereby replacing the actin speci¢c ADP-ribosyltransferase with the Rho-speci¢c C3. The treatment of cultured cells with C3 fusion toxin along with C2II results in a C3-speci¢c phenotype at more than a 300fold lower concentration of the fusion toxin than with C3 [16]. 2.2. Rho-glucosylating toxins Rho proteins are glucosylated by a group of toxins which are classi¢ed as LCCs because of their mass ( s 250 kDa) [17,18]. Members of this family are C. di¤- FEMSLE 9431 14-6-00 3 cile toxins A and B, which are the major virulence factors in antibiotic-associated diarrhoea and pseudomembranous colitis, the lethal and haemorrhagic toxins from Clostridium sordellii, and the K-toxin from Clostridium novyi, which may be involved in gas gangrene syndrome. In addition, several toxin variants (e.g. toxin B types 1470 and 8864), have been described which are functional toxin chimeras of toxin B and lethal toxin [19,20]. All these toxins consist of a single polypeptide chain harbouring a catalytic domain at their N-terminus (amino acid residues 1^546) and a cell-binding domain at their C-terminus [21]. The LCCs di¡er somewhat in their substrate and cosubstrate speci¢city. Toxins A and B, C. sordellii haemorrhagic toxin and C. novyi K-toxin modify all Rho GTPases studied so far; by contrast, lethal toxin and the toxin B type 1470 and 8864 modify Rac and possibly Cdc42, but not Rho [19,20]. In addition Ras subfamily proteins (Ras, Ral, Rap) are substrates for these toxins [22,23]. All large clostridial toxins accept UDP-glucose as a cosubstrate, with the exception of K-toxin which uses UDP-GlcNAc as a cosubstrate. The above mentioned LCCs modify Rho GTPases at a threonine residue (Thr37 of Rho, Thr35 of Rac/Cdc42) which is positioned within the switch-I region and is involved in nucleotide binding and co-ordination of the GTPase bound Mg2 ion. Mono-glucosylation of Thr37/35 has several important functional consequences for Rho GTPases. Most importantly, glucosylation inhibits the interaction of the GTPase with their e¡ectors [12,24]. Therefore glucosylated Rho is biologically inactive. In addition, glucosylation blocks the intrinsic and GAP-stimulated GTPase activity and inhibits nucleotide exchange by GEFs [12]. More recently, it was demonstrated that the glucosylation blocks the interaction of Rho with GDI, thereby preventing the cytosol membrane cycling [25]. 3. Toxins acting as Rho GAP proteins Rho GTPases are the target of `injected' toxins introduced into the eukaryotic target cells by a type-III secretion system, which depends on direct interaction between bacteria and the host cell. One group of injected toxins, including SptP, ExoS and YopE, are GAP-like proteins for Rho GTPases. The Salmonella typhimurium SptP (Salmonella protein tyrosine phosphatase) is a multidomain protein that possesses tyrosine phosphatase activity at its carboxy-terminal end [26]. The N-terminal part is similar to the N-terminus of ExoS and YopE. Recently SptP was shown to act as a GAP speci¢c for Cdc42 and Rac [27]. Also Pseudomonas aeruginosa exoenzyme S (ExoS) is a bifunctional and bimodular protein. Its C-terminus harbours an ADP-ribosyltransferase activity to modify Ras, thereby blocking the activation of Ras by GEFs [28]. The N-terminus of ExoS causes destruction of the actin cytoskeleton similar to known Rho-inactivating agents [29]. Cyaan Magenta Geel Zwart 4 M. Lerm et al. / FEMS Microbiology Letters 188 (2000) 1^6 Like SptP, this part of ExoS possesses GAP activity; however, in addition to Rac and Cdc42, it also targets Rho. Interestingly, the N-terminal domain of ExoS has only a slight homology to Rho GAPs, but shows homologies to Ras GAPs [30]. Recent crystal structure analysis of Rho and Ras GAPs showed that these enzymes stabilise the transition state of GTP hydrolysis by inserting an arginine ¢nger into the GTP-binding cavity of the GTPases [31]. In line with these ¢ndings, Arg146 of ExoS, which is equivalent to Arg209 of SptP, was shown to be essential for the GAP activity [30]. The third member of the GAP toxin family is the Yersinia protein YopE, which shares sequence homology with the GAP regions of SptP and ExoS, including the catalytically important arginine ¢nger. Recently, the GAP activity of YopE for Rho, Rac and Cdc42 was reported [32]. All these toxins comprise a novel group of bacterial protein toxins which are unique in acting on Rho GTPases in a non-covalent manner. 4. Rho-activating toxins In 1983, Caprioli et al. puri¢ed a toxin from E. coli strains isolated from enteritis-a¡ected children, which was termed CNF to indicate its necrotising activity on rabbit skin and cytotoxic e¡ects on cultured cells [33]. Later, a second CNF (designated CNF2) sharing 85% identical amino acids with CNF1 was identi¢ed [34]. CNFs are 115-kDa protein toxins with the catalytic domain located at the C-terminus (amino acid residues 709^ 1014) and the cell-binding domain at the N-terminus [35,36]. CNFs catalyse the deamidation of Gln63 of Rho (Gln61 of Rac and Cdc42) [37^39]. This enzyme activity removes the carboxamide nitrogen of Gln63, which is essential for the correct positioning of GTP for hydrolysis in the catalytic pocket of the GTPases. Thus, the toxins inhibit the intrinsic and GAP-stimulated GTPase activities and, thereby, block the switch-o¡ reaction of the GTPases. The resulting constitutive activation of Rho, Rac and Cdc42 explains the formation of stress ¢bres, membrane ru¥es and microspikes in CNF-treated cells. Another typical feature of CNF treatment is the formation of multinucleated giant cells, a phenomenon which results most likely from the inhibition of cytokinesis. The DNT produced by B. pertussis, B. parapertussis and B. bronchiseptica induces in cultured cells a similar phenotype as CNFs (e.g. stress ¢bre formation and multinucleation of giant cells). The toxin (V160 kDa) exhibits signi¢cant homology with CNFs at its C-terminus which harbours the catalytic domain [40]. Thus, similarly to CNFs, DNT deamidates Rho in vitro at Gln63 to inhibit its GTP-hydrolysing activity. Deamidation of Rho results in a slower migration of the GTPase in sodium dodecyl sulphate gel electrophoresis. Surprisingly, when Rho from DNT-treated cells was analysed, most of the protein exhibited faster migration than control Rho, but some mi- FEMSLE 9431 14-6-00 grated slower, suggesting a di¡erent type of modi¢cation than that induced by CNF [41]. The puzzle was solved when it was recognised that DNT possesses transglutaminase activity. DNT attaches primary amines onto Rho at position Gln63. Further comparison of the enzyme activities of DNT and CNFs revealed that both toxins are capable of catalysing deamidation and transglutamination reactions; however, DNT acts preferentially as a transglutaminase [36,42]. Recently it was reported that DNT uses various polyamines, e.g. spermidine and spermine, to transglutaminate Rho GTPases. Also, transglutamination blocks the GTPase activity and causes an increase in af¢nity for its e¡ector [43]. Rho proteins are also activated by the Salmonella protein SopE, which is delivered into the eukaryotic host cell by the type-III secretion system. SopE appears to be responsible for the membrane ru¥ing occurring concomitantly with host^cell interaction of Salmonella. Accordingly, it was shown that the Salmonella protein interacts with Rho GTPases to induce nucleotide exchange and, thereby, to activate several Rho GTPases including Rac and Cdc42 and, to a lesser extent, RhoA. Therefore, SopE acts like a GEF for Rho GTPases [44]. Intriguingly, through type-III system-mediated `injection' of toxins into the host cells, Salmonella a¡ects Rho GTPases and Rho-dependent signalling in a bidirectional manner. It causes activation of Rho GTPases by SopE to induce membrane ru¥ing and macropinocytosis, which ultimately results in cellular up-take of the bacteria and it turns o¡ the profound reorganisation of the actin cytoskeleton by terminating the active state of GTPase caused by the GAP-like protein SptP. Thus, in the case of Salmonella a dynamic manipulation of Rho signalling is achieved, which is not possible through covalent modi¢cation of Rho GTPases as caused by other toxins. 5. Conclusions Rho GTPases are targets of various bacterial protein toxins. The toxins are capable of either activating or inactivating the GTPases. Some toxins cause persistent and constitutive changes in the activity of Rho proteins, most likely resulting in `all or none' responses of the target cell. Other toxins (injected toxins) modulate the activity state of the GTPases in a rather dynamic manner, which is most likely time-, concentration- and space-dependent, resulting in a ¢ne tuning of cellular responses. From a teleological point of view, the reason for the targeting of Rho GTPases by bacterial toxins appears to be obvious. Rho proteins are master regulators in diverse signalling pathways including signalling in the immune surveillance system, in speci¢c and unspeci¢c host defence mechanisms. Inhibition or modulation of Rho GTPases by pathogens may help them to prevail. Therefore, precise knowledge of the actions of toxins is a prerequisite for understanding Cyaan Magenta Geel Zwart M. Lerm et al. / FEMS Microbiology Letters 188 (2000) 1^6 the pathogenesis of infectious diseases caused by the toxinproducing pathogens. 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