Download Bacterial protein toxins targeting Rho GTPases

FEMS Microbiology Letters 188 (2000) 1^6
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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
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Cyaan Magenta Geel Zwart
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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
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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.
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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¤-
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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
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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-
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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. Moreover, the elaboration of the
actions of toxins mentioned above uncovered their unique
speci¢city, selectivity and potency in acting on Rho
GTPases and made them useful tools for cell biological
studies to unravel the signalling pathways in which Rho
GTPases are involved.
[17]
[18]
[19]
References
[20]
[1] Van Aelst, L. and D'Souza-Schorey, C. (1997) Rho GTPases and
signaling networks. Genes Dev. 11, 2295^2322.
[2] Mackay, D.J.G. and Hall, A. (1998) Rho GTPases. J. Biol. Chem.
273, 20685^20688.
[3] Ridley, A. (2000) Rho. In: GTPases (Hall, A., Ed.), Vol. 24, pp. 89^
136. Oxford University Press, Oxford.
[4] Wei, Y., Zhang, Y., Derewenda, U., Liu, X., Minor, W., Nakamoto,
R.K., Somlyo, A.V., Somlyo, A.P. and Derewenda, Z.S. (1997) Crystal structure of RhoA-GDP and its functional implications. Nature
Struct. Biol. 4, 699^703.
[5] Hall, A. (1998) Rho GTPases and the actin cytoskeleton. Science 279,
509^514.
[6] Aktories, K., Weller, U. and Chhatwal, G.S. (1987) Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct
from botulinum C2 toxin. FEBS Lett. 212, 109^113.
[7] Braun, U., Habermann, B., Just, I., Aktories, K. and Vandekerckhove, J. (1989) Puri¢cation of the 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 from porcine brain cytosol and its
characterization as a GTP-binding protein highly homologous to the
rho gene product. FEBS Lett. 243, 70^76.
[8] Chardin, P., Boquet, P., Madaule, P., Popo¡, M.R., Rubin, E.J. and
Gill, D.M. (1989) The mammalian G protein rho C is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and a¡ects actin micro¢lament in Vero cells. EMBO J. 8, 1087^1092.
[9] Aktories, K., Barth, H., and Just, I. (2000) Clostridium botulinum C3
exoenzyme and C3-like transferases. In: Handbook of Experimental
Pharmacology, Vol. 145, pp. 207^233. Springer, Berlin.
[10] Just, I., Mohr, C., Schallehn, G., Menard, L., Didsbury, J.R., Vandekerckhove, J., van Damme, J. and Aktories, K. (1992) Puri¢cation
and characterization of an ADP-ribosyltransferase produced by Clostridium limosum. J. Biol. Chem. 267, 10274^10280.
[11] Sekine, A., Fujiwara, M. and Narumiya, S. (1989) Asparagine residue
in the rho gene product is the modi¢cation site for botulinum ADPribosyltransferase. J. Biol. Chem. 264, 8602^8605.
[12] Sehr, P., Joseph, G., Genth, H., Just, I., Pick, E. and Aktories, K.
(1998) Glucosylation and ADP-ribosylation of Rho proteins : e¡ects
on nucleotide binding, GTPase activity, and e¡ector-coupling. Biochemistry 37, 5296^5304.
[13] Takahashi, K., Sasaki, T., Mammoto, A., Takaishi, K., Kameyama,
T., Tsukita, S. and Takai, Y. (1997) Direct interaction of the Rho
GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein. J. Biol. Chem. 272, 23371^
23375.
[14] Fujihara, H., Walker, L.A., Gong, M.C., Lemichez, E., Boquet, P.,
Somlyo, A.V. and Somlyo, A.P. (1997) Inhibition of RhoA translocation and calcium sensitization by in vivo ADP-ribosylation with
the chimeric toxin DC3B. Mol. Biol. Cell 8, 2437^2447.
[15] Ohishi, I., Iwasaki, M. and Sakaguchi, G. (1980) Puri¢cation and
characterization of two components of botulinum C2 toxin. Infect.
Immun. 30, 668^673.
[16] Barth, H., Hofmann, F., Olenik, C., Just, I. and Aktories, K. (1998)
The N-terminal part of the enzyme component (C2I) of the binary
Clostridium botulinum C2 toxin interacts with the binding component
FEMSLE 9431 14-6-00
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
5
C2II and functions as a carrier system for a Rho ADP-ribosylating
C3-like fusion toxin. Infect. Immun. 66, 1364^1369.
Just, I., Selzer, J., Wilm, M., Von Eichel-Streiber, C., Mann, M. and
Aktories, K. (1995) Glucosylation of Rho proteins by Clostridium
di¤cile toxin B. Nature 375, 500^503.
Aktories, K. and Just, I. (1995) Monoglucosylation of low-molecularmass GTP-binding Rho proteins by clostridial cytotoxins. Trends
Cell Biol. 5, 441^443.
Soehn, F., Wagenknecht-Wiesner, A., Leukel, P., Kohl, M., Weidmann, M., Von Eichel-Streiber, C. and Braun, V. (1998) Genetic
rearrangements in the pathogenicity locus of Clostridium di¤cile
strain 8864: implications for transcription, expression and enzymatic
activity of toxins A and B. Mol. Gen. Genet. 258, 222^232.
Chaves-Olarte, E., Lo«w, P., Freer, E., Norlin, T., Weidmann, M.,
Von Eichel-Streiber, C. and Thelestam, M. (1999) A novel cytotoxin
from Clostridium di¤cile serogroup F is a functional hybrid between
two other large clostridial cytotoxins. J. Biol. Chem. 274, 11046^
11052.
Hofmann, F., Busch, C., Prepens, U., Just, I. and Aktories, K. (1997)
Localization of the glucosyltransferase activity of Clostridium di¤cile
toxin B to the N-terminal part of the holotoxin. J. Biol. Chem. 272,
11074^11078.
Just, I., Selzer, J., Hofmann, F., Green, G.A. and Aktories, K. (1996)
Inactivation of Ras by Clostridium sordellii lethal toxin-catalyzed
glucosylation. J. Biol. Chem. 271, 10149^10153.
Popo¡, M.R., Chaves, O.E., Lemichez, E., Von Eichel-Streiber, C.,
Thelestam, M., Chardin, P., Cussac, D., Chavrier, P., Flatau, G.,
Giry, M., Gunzburg, J. and Boquet, P. (1996) Ras, Rap, and Rac
small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation. J. Biol. Chem. 271, 10217^10224.
Herrmann, C., Ahmadian, M.R., Hofmann, F. and Just, I. (1998)
Functional consequences of monoglucosylation of H-Ras at e¡ector
domain amino acid threonine-35. J. Biol. Chem. 273, 16134^16139.
Genth, H., Aktories, K. and Just, I. (1999) Monoglucosylation of
RhoA at threonine-37 blocks cytosol-membrane cycling. J. Biol.
Chem. 274, 29050^29056.
Kaniga, K., Uralil, J., Bliska, J.B. and Galandrini, R. (1996) A secreted protein tyrosine phosphatase with modular e¡ector domains in
the bacterial pathogen Salmonella typhimurium. Mol. Microbiol. 21,
633^641.
Fu, Y. and Galän, J.E. (1999) A Salmonella protein antagonizes Rac1 and Cdc42 to mediate host-cell recovery after bacterial invasion.
Nature 401, 293^297.
Ganesan, A.K., Frank, D.W., Misra, R.P., Schmidt, G. and Barbieri,
J.T. (1998) Pseudomonas aeruginosa exoenzyme S ADP-ribosylates
Ras at multiple sites. J. Biol. Chem. 273, 7332^7337.
Pederson, K.J., Vallis, A.J., Aktories, K., Frank, D.W. and Barbieri,
J.T. (1999) The amino-terminal domain of Pseudomonas aeruginosa
ExoS disrupts actin ¢laments via small-molecular-weight GTP-binding proteins. Mol. Microbiol. 32, 393^401.
Goehring, U.-M., Schmidt, G., Pederson, K.J., Aktories, K. and
Barbieri, J.T. (1999) The N-terminal domain of Pseudomonas aeruginosa exoenzyme S is a GTPase-activating protein for Rho GTPases.
J. Biol. Chem. 274, 36369^36372.
Rittinger, K., Walker, P.A., Eccleston, J.F., Smerdon, S.J. and Gamblin, S.J. (1997) Structure at 1.65 A of RhoA and its GTPase-activating protein in complex with a transition-state analogue. Nature 389,
758^762.
von Pawel-Rammingen, U., Telepnev, M.V., Schmidt, G., Aktories,
K., Wolf-Watz, H. and Rosqvist, R. (2000) GAP activity of the
Yersinia YopE cytotoxin speci¢cally targets the Rho pathway : a
mechanism for disruption of actin micro¢lament structure. Mol. Microbiol., in press.
Caprioli, A., Falbo, V., Roda, L.G., Ruggeri, F.M. and Zona, C.
(1983) Partial puri¢cation and characterization of an Escherichia coli
toxic factor that induces morphological cell alterations. Infect. Immun. 39, 1300^1306.
Cyaan Magenta Geel Zwart
6
M. Lerm et al. / FEMS Microbiology Letters 188 (2000) 1^6
[34] de Rycke, J., Gonzälez, E.A., Blanco, J., Oswald, E., Blanco, M. and
Boivin, R. (1990) Evidence for two types of cytotoxic necrotising
factor in human and animal clinical isolates of Escherichia coli.
J. Clin. Microbiol. 28, 694^699.
[35] Lemichez, E., Flatau, G., Bruzzone, M., Boquet, P. and Gauthier, M.
(1997) Molecular localization of the Escherichia coli cytotoxic necrotizing factor CNF1 cell-binding and catalytic domains. Mol. Microbiol. 24, 1061^1070.
[36] Schmidt, G., Selzer, J., Lerm, M. and Aktories, K. (1998) The Rhodeamidating cytotoxic-necrotizing factor CNF1 from Escherichia coli
possesses transglutaminase activity: cysteine-866 and histidine-881
are essential for enzyme activity. J. Biol. Chem. 273, 13669^13674.
[37] Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Fiorentini, C. and Boquet, P. (1997) Toxin-induced activation of the G
protein p21 Rho by deamidation of glutamine. Nature 387, 729^733.
[38] Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M. and Aktories,
K. (1997) Gln63 of Rho is deamidated by Escherichia coli cytotoxic
necrotizing factor 1. Nature 387, 725^729.
[39] Lerm, M., Selzer, J., Ho¡meyer, A., Rapp, U.R., Aktories, K. and
Schmidt, G. (1998) Deamidation of Cdc42 and Rac by Escherichia
coli cytotoxic necrotizing factor 1 (CNF1): activation of c-Jun-Nterminal kinase in HeLa cells. Infect. Immun. 67, 496^503.
FEMSLE 9431 14-6-00
[40] Pullinger, G.D., Adams, T.E., Mullan, P.B., Garrod, T.I. and Lax,
A.J. (1996) Cloning, expression, and molecular characterization of
the dermonecrotic toxin gene of Bordetella spp.. Infect. Immun. 64,
4163^4171.
[41] Horiguchi, Y., Inoue, N., Masuda, M., Kashimoto, T., Katahira, J.,
Sugimoto, N. and Matsuda, M. (1997) Bordetella bronchiseptica dermonecrotizing toxin induces reorganization of actin stress ¢bers
through deamidation of Gln-63 of the GTP-binding protein Rho.
Proc. Natl. Acad. Sci. USA 94, 11623^11626.
[42] Schmidt, G., Goehring, U.-M., Schirmer, J., Lerm, M. and Aktories,
K. (1999) Identi¢cation of the C-terminal part of Bordetella dermonecrotic toxin (D-DNT) as a transglutaminase for Rho GTPases.
J. Biol. Chem. 274, 31875^31881.
[43] Masuda, M., Betancourt, L., Matsuzawa, T., Kashimoto, T., Takao,
T., Shimonishi, Y. and Horiguchi, Y. (2000) Activation of Rho
through a cross-link with polyamines catalyzed by Bordetella dermonecrotizing toxin. EMBO J. 19, 521^530.
[44] Hardt, W.-D., Chen, L.-M., Schuebel, K.E., Bustelo, X.R. and
Galän, J.E. (1998) S. typhimurium encodes an activator of Rho
GTPases that induces membrane ru¥ing and nuclear responses in
host cells. Cell 93, 815^826.
Cyaan Magenta Geel Zwart