Toxicon xxx (2015) 1e6
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Rho-modifying bacterial protein toxins from Photorhabdus species Thomas Jank a, Alexander E. Lang a, Klaus Aktories a, b, * a b
€t Freiburg, Albertstr. 25, 79104 Freiburg, Germany Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universita €t Freiburg, Freiburg, Germany Centre for Biological Signalling Studies (BIOSS), Albert-Ludwigs-Universita
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2015 Received in revised form 15 May 2015 Accepted 26 May 2015 Available online xxx
Photorhabdus bacteria live in symbiosis with entomopathogenic nematodes. The nematodes invade insect larvae, where they release the bacteria, which then produce toxins to kill the insects. Recently, the molecular mechanisms of some toxins from Photorhabdus luminescens and asymbiotica have been elucidated, showing that GTP-binding proteins of the Rho family are targets. The tripartite Tc toxin PTC5 from P. luminescens activates Rho proteins by ADP-ribosylation of a glutamine residue, which is involved in GTP hydrolysis, while PaTox from Photorhabdus asymbiotica inhibits the activity of GTPases by Nacetyl-glucosaminylation at tyrosine residues and activates Rho proteins indirectly by deamidation of heterotrimeric G proteins. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Glycosylation ADP-ribosylation Deamidation Heterotrimeric G proteins Actin Membrane binding
1. Introduction Many bacterial protein toxins, which act on mammalian organisms, affect target cells by modification of proteins of the Rho family. Rho proteins, which include ~20 proteins (best known are Rho, Rac and Cdc42 isoforms), are master regulators of the actin cytoskeleton and involved in numerous processes important for host protection against pathogens (Ridley and Hall, 1992; Jaffe and Hall, 2005; Aktories, 2011; Lemichez and Aktories, 2013; Popoff, 2014). Thus, Rho proteins are essentially involved in epithelial barrier function, phagocytosis, migration of immune cells, and immune cell signaling. Therefore, Rho proteins are preferred targets for bacterial toxins and effectors. The toxins inhibit or activate Rho protein functions by covalent modification of the switch proteins, including ADP-ribosylation (Aktories et al., 1989), glucosylation (Just et al., 1995a), AMPylation (adenylylation) (Yarbrough et al., 2009; Worby et al., 2009), deamidation (Schmidt et al., 1997; Flatau et al., 1997) and proteolytic cleavage (Shao et al., 2003). Bacterial toxins and effectors also modulate Rho protein function by mimicking the role of regulatory proteins like GTPaseactivating proteins (GAPs) and guanine nucleotide exchange factors
* Corresponding author. Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Albert-Ludwigs-Universit€ at Freiburg, Albertstr. 25, 79104 Freiburg, Germany. E-mail address: [email protected] (K. Aktories).
(GEFs) (Aktories, 2011). Mechanistic basis for the manipulation of Rho proteins by bacterial toxins/effectors is their switch functions in almost all eukaryotic cells (Ridley and Hall, 1992; Jaffe and Hall, 2005; Bustelo et al., 2007). Rho proteins belong to the Rassuperfamily of GTP-binding proteins (Madaule and Axel, 1985; Wennerberg et al., 2005). Accordingly, they are inactive in the GDP-bound form and active after nucleotide exchange induced by GEFs. In their active form, they interact with numerous effectors and, thereby, switch-on signaling and metabolic functions. The active state is terminated (switch-off) by hydrolysis of the bound GTP. This is caused by GAPs. Moreover, Rho proteins are regulated by GDIs (guanine nucleotide dissociation inhibitors), which keep the proteins in their inactive GTP-bound form in the cytosol (Jaffe and Hall, 2005; Wittinghofer and Vetter, 2011). 2. Photorhabdus bacteria Photorhabdus luminescens are Gram-negative bacteria, which live in the gut of entomopathogenic nematodes of the family Heterorhabditis. The nematodes invade insect larvae, where they release the bacteria by regurgitation (Waterfield et al., 2004, 2009; Ciche, 2007; Forst and Nealson, 1996). The bacteria produce a large array of different toxins, which eventually kill the larvae, thereby a food source for bacterial and nematodes is produced and bacteria as well as nematodes are able to propagate (ffrench-Constant et al., 2007). When the food source is exhausted, the bacteria are taken
http://dx.doi.org/10.1016/j.toxicon.2015.05.017 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jank, T., et al., Rho-modifying bacterial protein toxins from Photorhabdus species, Toxicon (2015), http:// dx.doi.org/10.1016/j.toxicon.2015.05.017
2
T. Jank et al. / Toxicon xxx (2015) 1e6
up by the nematodes that leave the cadaver of the larvae to invade a new insect. Therefore, nematodes carrying P. luminescens bacteria are used as biological insecticides (ffrench-Constant et al., 2007). At least three different species are known from the genus Photorhabdus: P. luminescens, P. temperata and P. asymbiotica (Waterfield et al., 2009). While P. temperata and P. luminescens are selective pathogens for insects, P. asymbiotica was shown to be a human pathogen as well (Peel et al., 1999; Gerrard et al., 2004). Moreover, it turned out that also P. asymbiotica is mutualistically associated with nematodes like the related species P. luminescens and temperata (Gerrard et al., 2004, 2006). The life cycle of nematodes and Photorhabdus species essentially depends on the production of protein toxins by the bacteria. Many types of Photorhabdus toxins have been reported (ffrench-Constant and Bowen, 2000). One of the first Photorhabdus toxins described in detail were the “makes caterpillar floppy” (MCF) toxins. The name refers to the phenotype of intoxicated insect larvae (Daborn et al., 2002) showing loss of insect body turgor. Other very potent toxins belong to a group of “toxin complex” (Tc) toxins, exhibiting masses of over 1.7 MDa (Waterfield et al., 2001; Bowen et al., 1998; Sheets et al., 2011; Gatsogiannis et al., 2013; Meusch et al., 2014). Tc toxins are released from the bacteria into the hemolymph of insect larvae, where they attack insect cells in an exotoxin like manner. Tc toxins are not only produced by insect-associated bacteria like Photorhabdus species, Serratia entomophila and Xenorhabdus nematophilus, genes encoding Tc toxin components are also found in the genome of Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica, which are important human pathogens (Heermann and Fuchs, 2008). Recent studies on Photorhabdus toxins showed that not only Rho proteins from vertebrate organisms but also insect Rho proteins are targets of bacterial protein toxins. These data are reviewed in the following sections.
closes the bottom of the pentameric shell (Meusch et al., 2014). The pore-forming region of TcA consists of a funnel formed by a region, which interacts with TcB and a large a-helical central channel. The linker connecting the shell and the channel acts as an entropic spring that drives the syringe-like injection of the TcA translocation channel into the membrane. The syringe/shell opens due to endosomal acidification (Fig. 1) (Gatsogiannis et al., 2013; Meusch et al., 2014). Interestingly, this happens also at high pH values (e.g. in the midgut of insects). TcB and TcC components are built by b-sheets that form together a large hollow cocoon which is closed by a distorted six-bladed bpropeller (Meusch et al., 2014), which is also the side where TcB interacts with TcA. The TcC component possesses an aspartyl autoprotease domain with two aspartates as a typical catalytic dyad. Previous studies were performed with the TcC isoforms TccC3 and TccC5, which both harbor ADP-ribosyltransferase domains at their C-terminus (Lang et al., 2010). It is suggested that the C-terminal ADP-ribosyltransferase domains are cleaved by the auto-protease and reside unfolded inside the cocoon of the TcC/TcB/TcA (PTC) toxin complexes. Cryo-EM studies suggest that the ADPribosyltransferase domains of TcC components pass through the b-propeller gate and enter the translocation pore of TcA (Meusch et al., 2014). The syringe-like injection of the TcA translocation channel into the membrane results in translocation of the ADPribosyltransferases into the cytosol of the target cell. Here, TccC3 ADP-ribosylates actin at threonine-148 (Lang et al., 2010). Threonine-148 is positioned at the actin-binding site of and prevents the binding of thymosin-b4 to actin. Because binding of thymosin-b4 to actin inhibits actin polymerization, ADPribosylation of threonine-148 induces actin polymerization (Fig. 1A) (Lang et al., 2010). The ADP-ribosyltransferase of TccC5 modifies Rho proteins.
3. Tc toxins
4. ADP-ribosylation of Rho proteins by TccC5 of Photorhabdus luminescens
Tc toxins consist of three components: TcA, TcB and TcC (complex is also termed PTC, Table 1), which each of them occurs in several isoforms (Bowen et al., 1998; ffrench-Constant and Waterfield, 2006; Gatsogiannis et al., 2013; Meusch et al., 2014). Recently, the structure of the complete 1.7 MDa Tc complex has been determined (Fig. 1) (Meusch et al., 2014). In this study, the Tc isoforms TcdA1 (TcA), TcdB2 (TcB) and TccC3/C5 (TcC) were studied. The TcA component forms a pentamer and is composed of eight domains: six domains forming the outer shell and two domains forming the translocation channel. The shell, which is connected by a ~48 amino acid linker to the domains forming the inner channel, is composed of a large extended a-helical domain, four putative receptor-binding domains and a neuraminidase-like domain that
Like TccC3, TccC5 causes death of larvae after injection of the whole toxin complex (PTC5) (Lang et al., 2010). In insect hemocytes and in HeLa cells, TccC5 induces polymerization of actin and formation of stress fibers. The combination of the actin-ADPribosylating toxins TccC3 and TccC5 enhances the effects on the cytoskeleton and causes a strong clustering of the actin cytoskeleton (Fig. 1A). Functional consequences are for example inhibition of phagocytosis of Escherichia coli particles by insect hemocytes. Sequence comparisons with other toxin ADP-ribosyltransferases and mutational analyses of TccC5 indicate that the Photorhabdus toxin belongs to the clostridial toxin-like ADP-ribosyltransferase (ARTC) subfamily of ADP-ribosyltransferases (Hottiger et al., 2010; Lang et al., 2010; Pfaumann et al., 2015). Typical for this
Table 1 Characterization of Photorhabdus toxins. Toxin/effector
Source
Structure
Toxin activity
Target (amino acid modified)
Functional consequences
PTC3
Photorhabdus luminescens Photorhabdus luminescens Photorhabdus asymbiotica
Tripartite toxin 5 TcdA1 (TcA) þ 1 TcdB2 (TcB) þ 1 TccC3 (TcC) Tripartite toxin 5 TcdA1 (TcA) þ 1 TcdB2 (TcB) þ 1 TccC5 (TcC) Single chain toxin
TccC3: ADP-ribosylation
Actin (Thr148)
Actin polymerization
TccC5: ADP-ribosylation
Rho GTPases (Gln61/63)
Rho activation
GlcNAcylation Deamidation
Rho GTPases (Tyr32/34) Heterotrimeric G proteins Gai, Gaq (Gln204/Gln209)
Rho inhibition Gai, Gaq activation
PTC5 PaTox
PTC3 and PTC5 from Photorhabdus luminescens have a tripartite structure and consist of TcA, TcB and TcC components. The enzyme components TccC3 and TccC5 possess ADPribosyltransferase activity and modify actin and Rho GTPases, respectively, resulting in actin polymerization and Rho activation. PaTox from Photorhabdus asymbiotica is a single chain toxin with at least two enzyme activities. The glycosyltransferase domain of PaTox causes attachment of N-acetylglucosamine (GlcNAc) onto Rho GTPases thereby inhibiting Rho proteins. The deamidation domain causes deamidation of heterotrimeric G proteins thereby activating the G proteins.
Please cite this article in press as: Jank, T., et al., Rho-modifying bacterial protein toxins from Photorhabdus species, Toxicon (2015), http:// dx.doi.org/10.1016/j.toxicon.2015.05.017
T. Jank et al. / Toxicon xxx (2015) 1e6
3
Fig. 1. Model of the structure and uptake of Photorhabdus luminescens Tc toxins. The holotoxin comprises TcA, TcB and TcC components. TcA consists of 8 domains; 6 domains form a shell and 2 domains a translocation pore. The toxin complex binds to receptors on the surface of host cells. Following endocytosis, low pH of endosomes lead to opening of the shell. The C-terminal regions of TcC proteins, which harbor ADP-ribosyltransferase activity in Tc isoforms TccC3 and TccC5 are then translocated through the TcA pore into the host cytoplasm. TccC3 ADP-ribosylates actin at threonine-148 thereby preventing the binding of the actin-sequestering protein thymosin-b4 to G-actin and favoring actin polymerization. TccC5 ADP-ribosylates Rho proteins at glutamine-63, thereby persistently activating Rho GTPases, which cause stress fiber formation and facilitate actin polymerization. Together, TccC3 and TccC5 cause clustering of F-actin (scheme modified from (Aktories et al., 2012) including data from (Gatsogiannis et al., 2013; Meusch et al., 2014)). Model of the holotoxin by fitting the TcdA1 and TcdB2-TccC3 crystal structure into the cryo-EM structure of the holotoxin complex obtained from (Meusch et al., 2014).
subfamily is an RSE motif with arginine and serine residues involved in NAD-binding and a so-called “catalytic” glutamate (Domenighini et al., 1994). In the case of TccC5, the RSE residues are Arg774, Ser809 and Glu886 (Pfaumann et al., 2015). Major protein substrates of TccC5-induced ADP-ribosylation are RhoA, B and C, Rac1-3 and Cdc42. A minor substrate is the Rho subfamily protein Tc10 (Pfaumann et al., 2015). Interestingly, also plant Rac-like proteins are at least in vitro substrates with a strong modification of Rop4. TccC5-catalyzed ADP-ribosylation of Rho proteins occurs at glutamine-63 and glutamine-61, respectively (Fig. 2) (Lang et al., 2010). Glutamine as an acceptor for ADPribosylating toxins is unique for TccC5. This residue (Gln61/63) is essential for GTP hydrolysis by Rho proteins (Wittinghofer and Vetter, 2011). ADP-ribosylation of Gln61/63 inhibits the turn-off mechanism of Rho proteins even in the presence of GAPs. This turns the molecular switches into a constitutively activated state (Lang et al., 2010; Aktories, 2011) and explains why TccC5 causes strong stress-fiber formation. Toxin-induced persistently activated
RhoA activates Rho kinase and formins, which cause actin polymerization and stress fiber formation (Matsui et al., 1996; Leung et al., 1996; Ishizaki et al., 1997; Lang et al., 2010; Pfaumann et al., 2015). In vitro assays showed that also Rac and Cdc42 are activated, which should result in lamellipodia and filopodia formation (Ridley et al., 1992; Nobes and Hall, 1995). Why the effect of RhoA activation is dominant remains to be clarified. Also cytotoxic necrotizing factors (CNFs) constitutively activate Rho proteins (Schmidt et al., 1997; Flatau et al., 1997; Hoffmann and Schmidt, 2004). These toxins, which are produced by E. coli and Yersinia species, deamidate glutamine-61/63 of Rho proteins resulting in glutamate at this position (Fig. 2). Change of the crucial glutamine residue to glutamate also prevents GTP hydrolysis by Rho proteins. Thus, ADP-ribosylation and deamidation cause similar results in term of the activation state of Rho proteins. However, precise analysis and comparison of the actions of TccC5 and CNFs revealed differences in intact cells. While the activation of RhoA protein by TccC5 in HeLa cells is persistent over several days,
Please cite this article in press as: Jank, T., et al., Rho-modifying bacterial protein toxins from Photorhabdus species, Toxicon (2015), http:// dx.doi.org/10.1016/j.toxicon.2015.05.017
4
T. Jank et al. / Toxicon xxx (2015) 1e6
6. PaTox GlcNAcylates Rho proteins at tyrosine 32/34
Fig. 2. Photorhabdus toxin catalyzed modification of Rho GTPases. Photorhabdus luminescens Tc-toxin complex with the enzyme component TccC5 ADP-ribosylates Rho GTPases (e.g. RhoA) at glutamine-61/63 rendering the GTPase constitutively active. This results in actin polymerization and increase in actin stress fiber formation. Note, at the same amino acid (Gln61/63), Rho proteins are activated by CNF toxin catalyzed deamidation of glutamine to glutamate. Photorhabdus asymbiotica toxin PaTox GlcNAcylates tyrosine-32/34 leading to a “switch off“ of the Rho GTPases, which results in actin disassembly and rounding-up of cells. Clostridial glucosylating toxins (CGTs) modify threonine-35/37, which also leads to an inactivation of Rho GTPases.
the activity state of RhoA after CNF1-treatment is increased for 2e4 h but returns to basal levels after 18 h, although the protein was still deamidated (Pfaumann et al., 2015). Similarly, the activity of Rac protein increases after CNF1 and TccC5 treatment for 2e6 h, whereas Rac activity decreases after 18 h in the case of CNF1, while it is stable with TccC5. Decrease of Rac activity induced by CNF1 is probably caused by proteolytic degradation of the activated protein (Doye et al., 2002; Lerm et al., 2002), a process, which is not observed with TccC5. Thus, both ADP-ribosylation and deamidation of Gln61/63 of Rho proteins cause activation of the GTPases; however, the time-dependent fate of the activation state is different. Similarly, functional and regulatory differences of Rho activation are observed in cell migration assays and in induction of MAL/SRF-dependent transcription after cell treatment with ADPribosylating or deamidating toxins (Pfaumann et al., 2015). These findings most likely reflect different cellular micro-environments, spatial restrictions and proteineprotein interactions after ADPribosylation or deamidation of Rho proteins. 5. Glycosylation of Rho proteins by PaTox Recently, a novel toxin produced by Photorhabdus asymbiotica, termed PaTox, was identified by sequence comparison with toxin glycosyltransferases (Jank et al., 2013) (Table 1). Especially high sequence similarity is observed with Clostridium difficile toxin B and the Legionella pneumophila effector Lgt1. The toxin has a mass of ~335 kDa and harbors a glycosyltransferase at its C-terminal part, which covers amino acid residues 2115 through 2449 (Fig. 3A). Microinjection of the holo-toxin into larvae of the greater wax moth (Galeria mellonella) causes death of the insect within 2e4 days. As found for other toxin glycosyltransferases, PaTox shares a DXD motif in its glycosyltransferase domain, which is essential for enzyme activity (see below) (Fig. 3B). When this motif is changed (e.g., exchange of DXD to NXN), the toxic effect of PaTox on larvae is strongly reduced. In cell culture (e.g., mammalian HeLa cells), PaTox causes destruction of the actin cytoskeleton and rounding up of cells, similar as found for C. difficile toxins A and B. Similar effects are also observed when only the glycosyltransferase domain is introduced into cultured cells. Again, using a DXD mutant of the glycosyltransferase domain (NXN-PaToxG) completely prevents the cytotoxic effects.
The glycosyltransferase domain of PaTox transfers N-acetylglucosamine (GlcNAc) from UDP-GlcNAc onto Rho proteins (Jank et al., 2013). In vitro, the toxin modifies RhoA, B and C, Rac proteins and Cdc42. Thus, PaTox shares the GlcNAcylation of Rho proteins with C. novyi a-toxin. However, while a-toxin like other clostridial glycosylating toxins modifies threonine-35/37 of RhoA, B and C, Rac and Cdc42, respectively (Just et al., 1995a, 1995b; Selzer et al., 1996; Popoff et al., 1996), PaTox GlcNAcylates tyrosine-32/34 of the GTPases respectively (Fig. 2) (Jank et al., 2013). This modification is unique for PaTox. NMR studies with GlcNAcylated RhoA revealed that PaTox like clostridial glycosylating toxins is a retaining glycosyltransferase, which attaches GlcNAc in an a-anomeric configuration onto Rho (Jank et al., 2013). This mode of sugar attachment is most likely of significance for the stability of the modified Rho protein, because intracellular glycohydrolases are known, which are able to split b-anomeric sugar-protein bonds but are not able to cleave a-anomeric sugar-protein linkages. Thus, PaTox induced modification of Rho is probably very stable. The functional consequences of the GlcNAcylation of Rho proteins are very similar to those observed after modification of the GTPases at threonine-35/37 by large clostridial glycosyltransferases (Jank et al., 2013; Sehr et al., 1998) (Fig. 2). These include (1) inhibition of effector interaction because the modification occurs in the effector region of the GTPases, (2) inhibition of activation of Rho proteins by GEFs, and, finally, blockade of the interaction of the GTPases with GAPs (Jank et al., 2013). However, while clostridial glycosyltransferase modify Rho proteins preferentially in the GDPbound form (Just et al., 1995a), PaTox catalyzed GlcNAcylation is favored with GTP-bound form of Rho GTPases. 7. Structure of PaTox glycosyltransferase and interaction with membranes The crystal structure of PaTox has been determined showing a GT-A type glycosyltransferase with the typical topology of this enzyme family. The catalytic site resembles the structure of large clostridial glycosyltransferases. This is not only true for the DXD motif, which is involved in divalent cation coordination but also for many conserved residues, which are important for UDP-GlcNAc binding including residues D2260, R2263 and Y2270. Active GTP-bound Rho proteins, which are the preferred substrates of PaTox are localized at the plasma membrane. This membrane attachment is achieved by the C-terminal isoprenylation of Rho proteins, which is in most cases of Rho GTPases a geranylgeranyl moiety (Fig. 3B) (Mohr et al., 1990; Adamson et al., 1992). Thus, also PaTox should be located at the membrane to meet its substrates. How is then the membrane attachment of PaTox achieved? Recent studies indicate that an N-terminal helical structure of PaTox is involved in membrane attachment (Jank et al., 2015). Basic residues of this subdomain interact with the negatively charged inner surface of the plasma membrane. Importantly, binding of PaTox to the cell membrane seems to be essential for its cytotoxicity. Exchange or deletion of the basic residues relocates PaTox to the cytosol, prevent modification of Rho, redistribution of the actin cytoskeleton and rounding-up of cells. When PaTox is brought back to the membrane by a lipid anchor, cell toxicity is restored. 8. The deamidase domain of PaTox Downstream of the glycosyltransferase of PaTox, a domain is located, which possesses deamidase activity (Fig. 3A). Sequence comparison shows significant similarity with SseI of a Salmonella
Please cite this article in press as: Jank, T., et al., Rho-modifying bacterial protein toxins from Photorhabdus species, Toxicon (2015), http:// dx.doi.org/10.1016/j.toxicon.2015.05.017
T. Jank et al. / Toxicon xxx (2015) 1e6
5
Fig. 3. Photorhabdus asymbiotica Toxin PaTox. (A) Domain organization of PaTox. (B) PaTox glycosyltransferase (PaToxG) associates with the negative charged inner leaflet of the plasma membrane (PM) with complementary charged residues on helix a1. Active Rho GTPases (e.g. RhoA) are also located at the PM by its C-terminal isoprenylation. At the PM PaTox glycosyltransferase modifies a conserved tyrosine residue within the switch I (swI) region (in case of RhoA Y34). The DXD motif in PaTox is a typical motif for GT-A type of glycosyltransferases and essential to coordinate a divalent metal ion and in turn the sugar donor UDP-GlcNAc.
Typhimurium effector protein of the type III secretion system SPI-2 (Bhaskaran and Stebbins, 2012) and PMT, a toxin from Pasteurella multocida, which was recognized to activate heterotrimeric G proteins by deamidation (Kitadokoro et al., 2007; Orth et al., 2009; Orth and Aktories, 2012). Similar as PMT, the deamidase domain of PaTox activates the a-subunits of the heterotrimeric Gai and Gaq proteins by deamidation of Gln204 and Gln209, respectively (Orth et al., 2013). Moreover, PaTox induced activation of Gaq causes activation of Rho proteins (e.g. RhoA), thereby the preferred substrate of the PaTox glycosyltransferase is generated. Thus, it has been suggested that a functional interplay between the deamidase and glycosyltransferase domains of PaTox exists. This view is supported by the findings that the membrane guiding caused by the Nterminal helix of the PaTox glycosyltransferase is also essential for the activation of the deamidase domain of PaTox on heterotrimeric G-proteins (Jank et al., 2015).
toxins and effectors employed by bacteria in host pathogen interaction. In fact, our laboratory has indications that also tyrosine glycosylation is not unique for PaTox and is utilized as mechanism to modify host target proteins of other hosts than insects. Ethical statement We declare that there are no ethical conflicts, because this is a review article. Acknowledgments Studies performed in the laboratory of the authors were financially supported by the Deutsche Forschungsgemeinschaft (DFG, project Ak6 22/2). Transparency document
9. Conclusion Rho GTPases are well established protein substrates of various bacterial protein toxins and effectors acting on mammalian cells. Recent studies indicate that Rho protein are similarly important substrates for the action of bacterial toxin in insects. Therefore, the same principles of toxin activity, molecular mechanisms and structural requirements for toxin-substrate interactions appear to apply also for these hostepathogen interactions. Nevertheless, significant differences in toxin targeting have been reported. For example, up to now the modification of tyrosine residues by glycosylation is unique for PaTox. However, one has to consider that we only know the tip of the iceberg of the huge armamentarium of
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2015.05.017. References Adamson, P., Marshall, C.J., Hall, A., Tilbrook, P.A., 1992. Post-translational modifications of p21rho proteins. J. Biol. Chem. 267, 20033e20038. Aktories, K., 2011. Bacterial protein toxins that modify host regulatory GTPases. Nat. Rev. Microbiol. 9, 487e498. € sener, S., Just, I., Hall, A., 1989. The rho gene product Aktories, K., Braun, U., Ro expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3. Biochem. Biophys. Res. Commun. 158, 209e213. Aktories, K., Schwan, C., Papatheodorou, P., Lang, A.E., 2012. Bidirectional attack on the actin cytoskeleton. Bacterial protein toxins causing polymerization or depolymerization of actin. Toxicon 60, 572e581.
Please cite this article in press as: Jank, T., et al., Rho-modifying bacterial protein toxins from Photorhabdus species, Toxicon (2015), http:// dx.doi.org/10.1016/j.toxicon.2015.05.017
6
T. Jank et al. / Toxicon xxx (2015) 1e6
Bhaskaran, S.S., Stebbins, C.E., 2012. Structure of the catalytic domain of the Salmonella virulence factor SseI. Acta Crystallogr. D. Biol. Crystallogr. 68, 1613e1621. Bowen, D., Rocheleau, T.A., Blackburn, M., Andreev, O., Golubeva, E., Bhartia, R., ffrench-Constant, R.H., 1998. Insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280, 2129e2132. Bustelo, X.R., Sauzeau, V., Berenjeno, I.M., 2007. GTP-binding proteins of the Rho/ Rac family: regulation, effectors and functions in vivo. Bioessays 29, 356e370. Ciche, T., 2007. The biology and genome of Heterorhabditis bacteriophora. WormBook 1e9. Daborn, P.J., Waterfield, N., Silva, C.P., Au, C.P.Y., Sharma, S., ffrench-Constant, R.H., 2002. A single photorhabdus gene, makes caterpillars floppy (mcf), allows Escherichia coli to persist within and kill insects. Proc. Natl. Acad. Sci. U. S. A. 99, 10742e10747. Domenighini, M., Magagnoli, C., Pizza, M., Rappuoli, R., 1994. Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins. Mol. Microbiol. 14, 41e50. ment, R., Buisson-Touati, C., Flatau, G., Doye, A., Mettouchi, A., Bossis, G., Cle Gagnoux, L., Piechaczyk, M., Boquet, P., Lemichez, E., 2002. CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. Cell 111, 553e564. ffrench-Constant, R., Waterfield, N., 2006. An ABC guide to the bacterial toxin complexes. Adv. Appl. Microbiol. 58, 169e183. ffrench-Constant, R.H., Bowen, D.J., 2000. Novel insecticidal toxins from nematodesymbiotic bacteria. Cell. Mol. Life Sci. 57, 828e833. ffrench-Constant, R.H., Dowling, A., Waterfield, N.R., 2007. Insecticidal toxins from Photorhabdus bacteria and their potential use in agriculture. Toxicon 49, 436e451. Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Fiorentini, C., Boquet, P., 1997. Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine. Nature 387, 729e733. Forst, S., Nealson, K., 1996. Molecular biology of the symbiotic-pathogenic bacteria Xenorhabdus spp. and Photorhabdus spp. Microbiol. Rev. 60, 21e43. Gatsogiannis, C., Lang, A.E., Meusch, D., Pfaumann, V., Hofnagel, O., Benz, R., Aktories, K., Raunser, S., 2013. A syringe-like injection mechanism in Photorhabdus luminescens toxins. Nature 495, 520e523. Gerrard, J., Waterfield, N., Vohra, R., ffrench-Constant, R., 2004. Human infection with Photorhabdus asymbiotica: an emerging bacterial pathogen. Microbes. Infect. 6, 229e237. Gerrard, J.G., Joyce, S.A., Clarke, D.J., ffrench-Constant, R.H., Nimmo, G.R., Looke, D.F., Feil, E.J., Pearce, L., Waterfield, N.R., 2006. Nematode symbiont for Photorhabdus asymbiotica. Emerg. Infect. Dis. 12, 1562e1564. Heermann, R., Fuchs, T.M., 2008. Comparative analysis of the Photorhabdus luminescens and the Yersinia enterocolitica genomes: uncovering candidate genes involved in insect pathogenicity. BMC Genomics 9, 40. Hoffmann, C., Schmidt, G., 2004. CNF and DNT. Rev. Physiol. Biochem. Pharmacol. 152, 49e63. Hottiger, M.O., Hassa, P.O., Luscher, B., Schuler, H., Koch-Nolte, F., 2010. Toward a unified nomenclature for mammalian ADP-ribosyltransferases. Trends Biochem. Sci. 35, 208e219. Ishizaki, T., Naito, M., Fujisawa, K., Maekawa, M., Watanabe, N., Saito, Y., Narumiya, S., 1997. p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions. FEBS Lett. 404, 118e124. Jaffe, A.B., Hall, A., 2005. Rho GTPases: biochemistry and biology. Annu. Rev. Cell. Dev. Biol. 21, 247e269. Jank, T., Bogdanovic, X., Wirth, C., Haaf, E., Spoerner, M., Bohmer, K.E., Steinemann, M., Orth, J.H., Kalbitzer, H.R., Warscheid, B., Hunte, C., Aktories, K., 2013. A bacterial toxin catalyzing tyrosine glycosylation of Rho and deamidation of Gq and Gi proteins. Nat. Struct. Mol. Biol. 20, 1273e1280. Jank, T., Trillhaase, C., Brozda, N., Steinemann, M., Schwan, C., Suss, R., Aktories, K., 2015. Intracellular plasma membrane guidance of Photorhabdus asymbiotica toxin is crucial for cell toxicity. FASEB J. pii: fj.14e269381. [Epub ahead of print]. Just, I., Selzer, J., Wilm, M., Von Eichel-Streiber, C., Mann, M., Aktories, K., 1995a. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375, 500e503. Just, I., Wilm, M., Selzer, J., Rex, G., Von Eichel-Streiber, C., Mann, M., Aktories, K., 1995b. The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins. J. Biol. Chem. 270, 13932e13936. Kitadokoro, K., Kamitani, S., Miyazawa, M., Hanajima-Ozawa, M., Fukui, A., Miyake, M., Horiguchi, Y., 2007. Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin. Proc. Natl. Acad. Sci. U. S. A. 104, 5139e5144. Lang, A.E., Schmidt, G., Schlosser, A., Hey, T.D., Larrinua, I.M., Sheets, J.J., Mannherz, H.G., Aktories, K., 2010. Photorhabdus luminescens toxins ADPribosylate actin and RhoA to force actin clustering. Science 327, 1139e1142. Lemichez, E., Aktories, K., 2013. Hijacking of Rho GTPases during bacterial infection. Exp. Cell Res. 319, 2329e2336. Lerm, M., Pop, M., Fritz, G., Aktories, K., Schmidt, G., 2002. Proteasomal degradation of cytotoxic necrotizing factor 1-activated Rac. Infect. Immun. 70, 4053e4058.
Leung, T., Chen, X.Q., Manser, E., Lim, L., 1996. The p160 RhoA-binding kinase ROKa is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol. Cell. Biol. 16, 5313e5327. Madaule, P., Axel, R., 1985. A novel ras-related gene family. Cell 41, 31e40. Matsui, T., Amano, M., Yamamoto, T., Chihara, K., Nakafuku, M., Ito, M., Nakano, T., Okawa, K., Iwamatsu, A., Kaibuchi, K., 1996. Rho-associated kinase, a novel serine threonine kinase, as a putative target for the small GTP binding protein Rho. EMBO J. 15, 2208e2216. Meusch, D., Gatsogiannis, C., Efremov, R.G., Lang, A.E., Hofnagel, O., Vetter, I.R., Aktories, K., Raunser, S., 2014. Mechanism of Tc toxin action revealed in molecular detail. Nature 508, 61e65. Mohr, C., Just, I., Hall, A., Aktories, K., 1990. Morphological alterations of Xenopus oocytes induced by valine- 14 p21rho depend on isoprenylation and are inhibited by Clostridium botulinum C3 ADP-ribosyltransferase. FEBS Lett. 275, 168e172. Nobes, C.D., Hall, A., 1995. Rho, rac and cdc42 GTPases: regulators of actin structures, cell adhesion and motility. Biochem. Soc. Trans. 23, 456e459. Orth, J.H., Aktories, K., 2012. Molecular biology of Pasteurella multocida toxin. Curr. Top. Microbiol. Immunol. 361, 73e92. Orth, J.H., Fester, I., Siegert, P., Weise, M., Lanner, U., Kamitani, S., Tachibana, T., Wilson, B.A., Schlosser, A., Horiguchi, Y., Aktories, K., 2013. Substrate specificity of Pasteurella multocida toxin for alpha subunits of heterotrimeric G proteins. FASEB J. 27, 832e842. Orth, J.H., Preuss, I., Fester, I., Schlosser, A., Wilson, B.A., Aktories, K., 2009. Pasteurella multocida toxin activation of heterotrimeric G proteins by deamidation. Proc. Natl. Acad. Sci. U. S. A. 106, 7179e7184. Peel, M.M., Alfredson, D.A., Gerrard, J.G., Davis, J.M., Robson, J.M., McDougall, R.J., Scullie, B.L., Akhurst, R.J., 1999. Isolation, identification, and molecular characterization of strains of Photorhabdus luminescens from infected humans in Australia. J. Clin. Microbiol. 37, 3647e3653. Pfaumann, V., Lang, A.E., Schwan, C., Schmidt, G., Aktories, K., 2015. The actin and Rho-modifying toxins PTC3 and PTC5 of Photorhabdus luminescens: enzyme characterization and induction of MAL/SRF-dependent transcription. Cell. Microbiol. 17, 579e594. Popoff, M.R., 2014. Bacterial factors exploit eukaryotic Rho GTPase signaling cascades to promote invasion and proliferation within their host. Small GTPases 5, e28209. Popoff, M.R., Chaves-Olarte, E., Lemichez, E., Von Eichel-Streiber, C., Thelestam, M., Chardin, P., Cussac, D., Antonny, B., Chavrier, P., Flatau, G., Giry, M., de, G.J., Boquet, P., 1996. Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation. J. Biol. Chem. 271, 10217e10224. Ridley, A.J., Hall, A., 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70, 389e399. Ridley, A.J., Paterson, H.F., Johnston, C.L., Diekmann, D., Hall, A., 1992. The small GTPbinding protein rac regulates growth factor- induced membrane ruffling. Cell 70, 401e410. Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M., Aktories, K., 1997. Gln63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor 1. Nature 387, 725e729. Sehr, P., Joseph, G., Genth, H., Just, I., Pick, E., Aktories, K., 1998. Glucosylation and ADP-ribosylation of Rho proteins e effects on nucleotide binding, GTPase activity, and effector-coupling. Biochemistry 37, 5296e5304. Selzer, J., Hofmann, F., Rex, G., Wilm, M., Mann, M., Just, I., Aktories, K., 1996. Clostridium novyi a-toxin-catalyzed incorporation of GlcNAc into Rho subfamily proteins. J. Biol. Chem. 271, 25173e25177. Shao, F., Vacratsis, P.O., Bao, Z., Bowers, K.E., Fierke, C.A., Dixon, J.E., 2003. Biochemical characterization of the Yersinia YopT protease: cleavage site and recognition elements in Rho GTPases. Proc. Natl. Acad. Sci. U. S. A. 100, 904e909. Sheets, J.J., Hey, T.D., Fencil, K.J., Burton, S.L., Ni, W., Lang, A.E., Benz, R., Aktories, K., 2011. Insecticidal toxin complex proteins from Xenorhabdus nematophilus: structure and pore formation. J. Biol. Chem. 286, 22742e22749. Waterfield, N.R., Bowen, D.J., Fetherston, J.D., Perry, R.D., ffrench-Constant, R.H., 2001. The tc genes of Photorhabdus: a growing family. Trends Microbiol. 9, 185e191. Waterfield, N.R., Ciche, T., Clarke, D., 2009. Photorhabdus and a host of hosts. Annu. Rev. Microbiol. 63, 557e574. Waterfield, N.R., Wren, B.W., ffrench-Constant, R.H., 2004. Invertebrates as a source of emerging human pathogens. Nat. Rev. Microbiol. 2, 833e841. Wennerberg, K., Rossman, K.L., Der, C.J., 2005. The Ras superfamily at a glance. J. Cell. Sci. 118, 843e846. Wittinghofer, A., Vetter, I.R., 2011. Structure-function relationships of the G domain, a canonical switch motif. Annu. Rev. Biochem. 80, 943e971. Worby, C.A., Mattoo, S., Kruger, R.P., Corbeil, L.B., Koller, A., Mendez, J.C., Zekarias, B., Lazar, C., Dixon, J.E., 2009. The fic domain: regulation of cell signaling by adenylylation. Mol. Cell. 34, 93e103. Yarbrough, M.L., Li, Y., Kinch, L.N., Grishin, N.V., Ball, H.L., Orth, K., 2009. AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling. Science 323, 269e272.
Please cite this article in press as: Jank, T., et al., Rho-modifying bacterial protein toxins from Photorhabdus species, Toxicon (2015), http:// dx.doi.org/10.1016/j.toxicon.2015.05.017
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Rho-modifying bacterial protein toxins from Photorhabdus species Thomas Jank a, Alexander E. Lang a, Klaus Aktories a, b, * a b
€t Freiburg, Albertstr. 25, 79104 Freiburg, Germany Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universita €t Freiburg, Freiburg, Germany Centre for Biological Signalling Studies (BIOSS), Albert-Ludwigs-Universita
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2015 Received in revised form 15 May 2015 Accepted 26 May 2015 Available online xxx
Photorhabdus bacteria live in symbiosis with entomopathogenic nematodes. The nematodes invade insect larvae, where they release the bacteria, which then produce toxins to kill the insects. Recently, the molecular mechanisms of some toxins from Photorhabdus luminescens and asymbiotica have been elucidated, showing that GTP-binding proteins of the Rho family are targets. The tripartite Tc toxin PTC5 from P. luminescens activates Rho proteins by ADP-ribosylation of a glutamine residue, which is involved in GTP hydrolysis, while PaTox from Photorhabdus asymbiotica inhibits the activity of GTPases by Nacetyl-glucosaminylation at tyrosine residues and activates Rho proteins indirectly by deamidation of heterotrimeric G proteins. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Glycosylation ADP-ribosylation Deamidation Heterotrimeric G proteins Actin Membrane binding
1. Introduction Many bacterial protein toxins, which act on mammalian organisms, affect target cells by modification of proteins of the Rho family. Rho proteins, which include ~20 proteins (best known are Rho, Rac and Cdc42 isoforms), are master regulators of the actin cytoskeleton and involved in numerous processes important for host protection against pathogens (Ridley and Hall, 1992; Jaffe and Hall, 2005; Aktories, 2011; Lemichez and Aktories, 2013; Popoff, 2014). Thus, Rho proteins are essentially involved in epithelial barrier function, phagocytosis, migration of immune cells, and immune cell signaling. Therefore, Rho proteins are preferred targets for bacterial toxins and effectors. The toxins inhibit or activate Rho protein functions by covalent modification of the switch proteins, including ADP-ribosylation (Aktories et al., 1989), glucosylation (Just et al., 1995a), AMPylation (adenylylation) (Yarbrough et al., 2009; Worby et al., 2009), deamidation (Schmidt et al., 1997; Flatau et al., 1997) and proteolytic cleavage (Shao et al., 2003). Bacterial toxins and effectors also modulate Rho protein function by mimicking the role of regulatory proteins like GTPaseactivating proteins (GAPs) and guanine nucleotide exchange factors
* Corresponding author. Institut für Experimentelle und Klinische Pharmakologie und Toxikologie Albert-Ludwigs-Universit€ at Freiburg, Albertstr. 25, 79104 Freiburg, Germany. E-mail address: [email protected] (K. Aktories).
(GEFs) (Aktories, 2011). Mechanistic basis for the manipulation of Rho proteins by bacterial toxins/effectors is their switch functions in almost all eukaryotic cells (Ridley and Hall, 1992; Jaffe and Hall, 2005; Bustelo et al., 2007). Rho proteins belong to the Rassuperfamily of GTP-binding proteins (Madaule and Axel, 1985; Wennerberg et al., 2005). Accordingly, they are inactive in the GDP-bound form and active after nucleotide exchange induced by GEFs. In their active form, they interact with numerous effectors and, thereby, switch-on signaling and metabolic functions. The active state is terminated (switch-off) by hydrolysis of the bound GTP. This is caused by GAPs. Moreover, Rho proteins are regulated by GDIs (guanine nucleotide dissociation inhibitors), which keep the proteins in their inactive GTP-bound form in the cytosol (Jaffe and Hall, 2005; Wittinghofer and Vetter, 2011). 2. Photorhabdus bacteria Photorhabdus luminescens are Gram-negative bacteria, which live in the gut of entomopathogenic nematodes of the family Heterorhabditis. The nematodes invade insect larvae, where they release the bacteria by regurgitation (Waterfield et al., 2004, 2009; Ciche, 2007; Forst and Nealson, 1996). The bacteria produce a large array of different toxins, which eventually kill the larvae, thereby a food source for bacterial and nematodes is produced and bacteria as well as nematodes are able to propagate (ffrench-Constant et al., 2007). When the food source is exhausted, the bacteria are taken
http://dx.doi.org/10.1016/j.toxicon.2015.05.017 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jank, T., et al., Rho-modifying bacterial protein toxins from Photorhabdus species, Toxicon (2015), http:// dx.doi.org/10.1016/j.toxicon.2015.05.017
2
T. Jank et al. / Toxicon xxx (2015) 1e6
up by the nematodes that leave the cadaver of the larvae to invade a new insect. Therefore, nematodes carrying P. luminescens bacteria are used as biological insecticides (ffrench-Constant et al., 2007). At least three different species are known from the genus Photorhabdus: P. luminescens, P. temperata and P. asymbiotica (Waterfield et al., 2009). While P. temperata and P. luminescens are selective pathogens for insects, P. asymbiotica was shown to be a human pathogen as well (Peel et al., 1999; Gerrard et al., 2004). Moreover, it turned out that also P. asymbiotica is mutualistically associated with nematodes like the related species P. luminescens and temperata (Gerrard et al., 2004, 2006). The life cycle of nematodes and Photorhabdus species essentially depends on the production of protein toxins by the bacteria. Many types of Photorhabdus toxins have been reported (ffrench-Constant and Bowen, 2000). One of the first Photorhabdus toxins described in detail were the “makes caterpillar floppy” (MCF) toxins. The name refers to the phenotype of intoxicated insect larvae (Daborn et al., 2002) showing loss of insect body turgor. Other very potent toxins belong to a group of “toxin complex” (Tc) toxins, exhibiting masses of over 1.7 MDa (Waterfield et al., 2001; Bowen et al., 1998; Sheets et al., 2011; Gatsogiannis et al., 2013; Meusch et al., 2014). Tc toxins are released from the bacteria into the hemolymph of insect larvae, where they attack insect cells in an exotoxin like manner. Tc toxins are not only produced by insect-associated bacteria like Photorhabdus species, Serratia entomophila and Xenorhabdus nematophilus, genes encoding Tc toxin components are also found in the genome of Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica, which are important human pathogens (Heermann and Fuchs, 2008). Recent studies on Photorhabdus toxins showed that not only Rho proteins from vertebrate organisms but also insect Rho proteins are targets of bacterial protein toxins. These data are reviewed in the following sections.
closes the bottom of the pentameric shell (Meusch et al., 2014). The pore-forming region of TcA consists of a funnel formed by a region, which interacts with TcB and a large a-helical central channel. The linker connecting the shell and the channel acts as an entropic spring that drives the syringe-like injection of the TcA translocation channel into the membrane. The syringe/shell opens due to endosomal acidification (Fig. 1) (Gatsogiannis et al., 2013; Meusch et al., 2014). Interestingly, this happens also at high pH values (e.g. in the midgut of insects). TcB and TcC components are built by b-sheets that form together a large hollow cocoon which is closed by a distorted six-bladed bpropeller (Meusch et al., 2014), which is also the side where TcB interacts with TcA. The TcC component possesses an aspartyl autoprotease domain with two aspartates as a typical catalytic dyad. Previous studies were performed with the TcC isoforms TccC3 and TccC5, which both harbor ADP-ribosyltransferase domains at their C-terminus (Lang et al., 2010). It is suggested that the C-terminal ADP-ribosyltransferase domains are cleaved by the auto-protease and reside unfolded inside the cocoon of the TcC/TcB/TcA (PTC) toxin complexes. Cryo-EM studies suggest that the ADPribosyltransferase domains of TcC components pass through the b-propeller gate and enter the translocation pore of TcA (Meusch et al., 2014). The syringe-like injection of the TcA translocation channel into the membrane results in translocation of the ADPribosyltransferases into the cytosol of the target cell. Here, TccC3 ADP-ribosylates actin at threonine-148 (Lang et al., 2010). Threonine-148 is positioned at the actin-binding site of and prevents the binding of thymosin-b4 to actin. Because binding of thymosin-b4 to actin inhibits actin polymerization, ADPribosylation of threonine-148 induces actin polymerization (Fig. 1A) (Lang et al., 2010). The ADP-ribosyltransferase of TccC5 modifies Rho proteins.
3. Tc toxins
4. ADP-ribosylation of Rho proteins by TccC5 of Photorhabdus luminescens
Tc toxins consist of three components: TcA, TcB and TcC (complex is also termed PTC, Table 1), which each of them occurs in several isoforms (Bowen et al., 1998; ffrench-Constant and Waterfield, 2006; Gatsogiannis et al., 2013; Meusch et al., 2014). Recently, the structure of the complete 1.7 MDa Tc complex has been determined (Fig. 1) (Meusch et al., 2014). In this study, the Tc isoforms TcdA1 (TcA), TcdB2 (TcB) and TccC3/C5 (TcC) were studied. The TcA component forms a pentamer and is composed of eight domains: six domains forming the outer shell and two domains forming the translocation channel. The shell, which is connected by a ~48 amino acid linker to the domains forming the inner channel, is composed of a large extended a-helical domain, four putative receptor-binding domains and a neuraminidase-like domain that
Like TccC3, TccC5 causes death of larvae after injection of the whole toxin complex (PTC5) (Lang et al., 2010). In insect hemocytes and in HeLa cells, TccC5 induces polymerization of actin and formation of stress fibers. The combination of the actin-ADPribosylating toxins TccC3 and TccC5 enhances the effects on the cytoskeleton and causes a strong clustering of the actin cytoskeleton (Fig. 1A). Functional consequences are for example inhibition of phagocytosis of Escherichia coli particles by insect hemocytes. Sequence comparisons with other toxin ADP-ribosyltransferases and mutational analyses of TccC5 indicate that the Photorhabdus toxin belongs to the clostridial toxin-like ADP-ribosyltransferase (ARTC) subfamily of ADP-ribosyltransferases (Hottiger et al., 2010; Lang et al., 2010; Pfaumann et al., 2015). Typical for this
Table 1 Characterization of Photorhabdus toxins. Toxin/effector
Source
Structure
Toxin activity
Target (amino acid modified)
Functional consequences
PTC3
Photorhabdus luminescens Photorhabdus luminescens Photorhabdus asymbiotica
Tripartite toxin 5 TcdA1 (TcA) þ 1 TcdB2 (TcB) þ 1 TccC3 (TcC) Tripartite toxin 5 TcdA1 (TcA) þ 1 TcdB2 (TcB) þ 1 TccC5 (TcC) Single chain toxin
TccC3: ADP-ribosylation
Actin (Thr148)
Actin polymerization
TccC5: ADP-ribosylation
Rho GTPases (Gln61/63)
Rho activation
GlcNAcylation Deamidation
Rho GTPases (Tyr32/34) Heterotrimeric G proteins Gai, Gaq (Gln204/Gln209)
Rho inhibition Gai, Gaq activation
PTC5 PaTox
PTC3 and PTC5 from Photorhabdus luminescens have a tripartite structure and consist of TcA, TcB and TcC components. The enzyme components TccC3 and TccC5 possess ADPribosyltransferase activity and modify actin and Rho GTPases, respectively, resulting in actin polymerization and Rho activation. PaTox from Photorhabdus asymbiotica is a single chain toxin with at least two enzyme activities. The glycosyltransferase domain of PaTox causes attachment of N-acetylglucosamine (GlcNAc) onto Rho GTPases thereby inhibiting Rho proteins. The deamidation domain causes deamidation of heterotrimeric G proteins thereby activating the G proteins.
Please cite this article in press as: Jank, T., et al., Rho-modifying bacterial protein toxins from Photorhabdus species, Toxicon (2015), http:// dx.doi.org/10.1016/j.toxicon.2015.05.017
T. Jank et al. / Toxicon xxx (2015) 1e6
3
Fig. 1. Model of the structure and uptake of Photorhabdus luminescens Tc toxins. The holotoxin comprises TcA, TcB and TcC components. TcA consists of 8 domains; 6 domains form a shell and 2 domains a translocation pore. The toxin complex binds to receptors on the surface of host cells. Following endocytosis, low pH of endosomes lead to opening of the shell. The C-terminal regions of TcC proteins, which harbor ADP-ribosyltransferase activity in Tc isoforms TccC3 and TccC5 are then translocated through the TcA pore into the host cytoplasm. TccC3 ADP-ribosylates actin at threonine-148 thereby preventing the binding of the actin-sequestering protein thymosin-b4 to G-actin and favoring actin polymerization. TccC5 ADP-ribosylates Rho proteins at glutamine-63, thereby persistently activating Rho GTPases, which cause stress fiber formation and facilitate actin polymerization. Together, TccC3 and TccC5 cause clustering of F-actin (scheme modified from (Aktories et al., 2012) including data from (Gatsogiannis et al., 2013; Meusch et al., 2014)). Model of the holotoxin by fitting the TcdA1 and TcdB2-TccC3 crystal structure into the cryo-EM structure of the holotoxin complex obtained from (Meusch et al., 2014).
subfamily is an RSE motif with arginine and serine residues involved in NAD-binding and a so-called “catalytic” glutamate (Domenighini et al., 1994). In the case of TccC5, the RSE residues are Arg774, Ser809 and Glu886 (Pfaumann et al., 2015). Major protein substrates of TccC5-induced ADP-ribosylation are RhoA, B and C, Rac1-3 and Cdc42. A minor substrate is the Rho subfamily protein Tc10 (Pfaumann et al., 2015). Interestingly, also plant Rac-like proteins are at least in vitro substrates with a strong modification of Rop4. TccC5-catalyzed ADP-ribosylation of Rho proteins occurs at glutamine-63 and glutamine-61, respectively (Fig. 2) (Lang et al., 2010). Glutamine as an acceptor for ADPribosylating toxins is unique for TccC5. This residue (Gln61/63) is essential for GTP hydrolysis by Rho proteins (Wittinghofer and Vetter, 2011). ADP-ribosylation of Gln61/63 inhibits the turn-off mechanism of Rho proteins even in the presence of GAPs. This turns the molecular switches into a constitutively activated state (Lang et al., 2010; Aktories, 2011) and explains why TccC5 causes strong stress-fiber formation. Toxin-induced persistently activated
RhoA activates Rho kinase and formins, which cause actin polymerization and stress fiber formation (Matsui et al., 1996; Leung et al., 1996; Ishizaki et al., 1997; Lang et al., 2010; Pfaumann et al., 2015). In vitro assays showed that also Rac and Cdc42 are activated, which should result in lamellipodia and filopodia formation (Ridley et al., 1992; Nobes and Hall, 1995). Why the effect of RhoA activation is dominant remains to be clarified. Also cytotoxic necrotizing factors (CNFs) constitutively activate Rho proteins (Schmidt et al., 1997; Flatau et al., 1997; Hoffmann and Schmidt, 2004). These toxins, which are produced by E. coli and Yersinia species, deamidate glutamine-61/63 of Rho proteins resulting in glutamate at this position (Fig. 2). Change of the crucial glutamine residue to glutamate also prevents GTP hydrolysis by Rho proteins. Thus, ADP-ribosylation and deamidation cause similar results in term of the activation state of Rho proteins. However, precise analysis and comparison of the actions of TccC5 and CNFs revealed differences in intact cells. While the activation of RhoA protein by TccC5 in HeLa cells is persistent over several days,
Please cite this article in press as: Jank, T., et al., Rho-modifying bacterial protein toxins from Photorhabdus species, Toxicon (2015), http:// dx.doi.org/10.1016/j.toxicon.2015.05.017
4
T. Jank et al. / Toxicon xxx (2015) 1e6
6. PaTox GlcNAcylates Rho proteins at tyrosine 32/34
Fig. 2. Photorhabdus toxin catalyzed modification of Rho GTPases. Photorhabdus luminescens Tc-toxin complex with the enzyme component TccC5 ADP-ribosylates Rho GTPases (e.g. RhoA) at glutamine-61/63 rendering the GTPase constitutively active. This results in actin polymerization and increase in actin stress fiber formation. Note, at the same amino acid (Gln61/63), Rho proteins are activated by CNF toxin catalyzed deamidation of glutamine to glutamate. Photorhabdus asymbiotica toxin PaTox GlcNAcylates tyrosine-32/34 leading to a “switch off“ of the Rho GTPases, which results in actin disassembly and rounding-up of cells. Clostridial glucosylating toxins (CGTs) modify threonine-35/37, which also leads to an inactivation of Rho GTPases.
the activity state of RhoA after CNF1-treatment is increased for 2e4 h but returns to basal levels after 18 h, although the protein was still deamidated (Pfaumann et al., 2015). Similarly, the activity of Rac protein increases after CNF1 and TccC5 treatment for 2e6 h, whereas Rac activity decreases after 18 h in the case of CNF1, while it is stable with TccC5. Decrease of Rac activity induced by CNF1 is probably caused by proteolytic degradation of the activated protein (Doye et al., 2002; Lerm et al., 2002), a process, which is not observed with TccC5. Thus, both ADP-ribosylation and deamidation of Gln61/63 of Rho proteins cause activation of the GTPases; however, the time-dependent fate of the activation state is different. Similarly, functional and regulatory differences of Rho activation are observed in cell migration assays and in induction of MAL/SRF-dependent transcription after cell treatment with ADPribosylating or deamidating toxins (Pfaumann et al., 2015). These findings most likely reflect different cellular micro-environments, spatial restrictions and proteineprotein interactions after ADPribosylation or deamidation of Rho proteins. 5. Glycosylation of Rho proteins by PaTox Recently, a novel toxin produced by Photorhabdus asymbiotica, termed PaTox, was identified by sequence comparison with toxin glycosyltransferases (Jank et al., 2013) (Table 1). Especially high sequence similarity is observed with Clostridium difficile toxin B and the Legionella pneumophila effector Lgt1. The toxin has a mass of ~335 kDa and harbors a glycosyltransferase at its C-terminal part, which covers amino acid residues 2115 through 2449 (Fig. 3A). Microinjection of the holo-toxin into larvae of the greater wax moth (Galeria mellonella) causes death of the insect within 2e4 days. As found for other toxin glycosyltransferases, PaTox shares a DXD motif in its glycosyltransferase domain, which is essential for enzyme activity (see below) (Fig. 3B). When this motif is changed (e.g., exchange of DXD to NXN), the toxic effect of PaTox on larvae is strongly reduced. In cell culture (e.g., mammalian HeLa cells), PaTox causes destruction of the actin cytoskeleton and rounding up of cells, similar as found for C. difficile toxins A and B. Similar effects are also observed when only the glycosyltransferase domain is introduced into cultured cells. Again, using a DXD mutant of the glycosyltransferase domain (NXN-PaToxG) completely prevents the cytotoxic effects.
The glycosyltransferase domain of PaTox transfers N-acetylglucosamine (GlcNAc) from UDP-GlcNAc onto Rho proteins (Jank et al., 2013). In vitro, the toxin modifies RhoA, B and C, Rac proteins and Cdc42. Thus, PaTox shares the GlcNAcylation of Rho proteins with C. novyi a-toxin. However, while a-toxin like other clostridial glycosylating toxins modifies threonine-35/37 of RhoA, B and C, Rac and Cdc42, respectively (Just et al., 1995a, 1995b; Selzer et al., 1996; Popoff et al., 1996), PaTox GlcNAcylates tyrosine-32/34 of the GTPases respectively (Fig. 2) (Jank et al., 2013). This modification is unique for PaTox. NMR studies with GlcNAcylated RhoA revealed that PaTox like clostridial glycosylating toxins is a retaining glycosyltransferase, which attaches GlcNAc in an a-anomeric configuration onto Rho (Jank et al., 2013). This mode of sugar attachment is most likely of significance for the stability of the modified Rho protein, because intracellular glycohydrolases are known, which are able to split b-anomeric sugar-protein bonds but are not able to cleave a-anomeric sugar-protein linkages. Thus, PaTox induced modification of Rho is probably very stable. The functional consequences of the GlcNAcylation of Rho proteins are very similar to those observed after modification of the GTPases at threonine-35/37 by large clostridial glycosyltransferases (Jank et al., 2013; Sehr et al., 1998) (Fig. 2). These include (1) inhibition of effector interaction because the modification occurs in the effector region of the GTPases, (2) inhibition of activation of Rho proteins by GEFs, and, finally, blockade of the interaction of the GTPases with GAPs (Jank et al., 2013). However, while clostridial glycosyltransferase modify Rho proteins preferentially in the GDPbound form (Just et al., 1995a), PaTox catalyzed GlcNAcylation is favored with GTP-bound form of Rho GTPases. 7. Structure of PaTox glycosyltransferase and interaction with membranes The crystal structure of PaTox has been determined showing a GT-A type glycosyltransferase with the typical topology of this enzyme family. The catalytic site resembles the structure of large clostridial glycosyltransferases. This is not only true for the DXD motif, which is involved in divalent cation coordination but also for many conserved residues, which are important for UDP-GlcNAc binding including residues D2260, R2263 and Y2270. Active GTP-bound Rho proteins, which are the preferred substrates of PaTox are localized at the plasma membrane. This membrane attachment is achieved by the C-terminal isoprenylation of Rho proteins, which is in most cases of Rho GTPases a geranylgeranyl moiety (Fig. 3B) (Mohr et al., 1990; Adamson et al., 1992). Thus, also PaTox should be located at the membrane to meet its substrates. How is then the membrane attachment of PaTox achieved? Recent studies indicate that an N-terminal helical structure of PaTox is involved in membrane attachment (Jank et al., 2015). Basic residues of this subdomain interact with the negatively charged inner surface of the plasma membrane. Importantly, binding of PaTox to the cell membrane seems to be essential for its cytotoxicity. Exchange or deletion of the basic residues relocates PaTox to the cytosol, prevent modification of Rho, redistribution of the actin cytoskeleton and rounding-up of cells. When PaTox is brought back to the membrane by a lipid anchor, cell toxicity is restored. 8. The deamidase domain of PaTox Downstream of the glycosyltransferase of PaTox, a domain is located, which possesses deamidase activity (Fig. 3A). Sequence comparison shows significant similarity with SseI of a Salmonella
Please cite this article in press as: Jank, T., et al., Rho-modifying bacterial protein toxins from Photorhabdus species, Toxicon (2015), http:// dx.doi.org/10.1016/j.toxicon.2015.05.017
T. Jank et al. / Toxicon xxx (2015) 1e6
5
Fig. 3. Photorhabdus asymbiotica Toxin PaTox. (A) Domain organization of PaTox. (B) PaTox glycosyltransferase (PaToxG) associates with the negative charged inner leaflet of the plasma membrane (PM) with complementary charged residues on helix a1. Active Rho GTPases (e.g. RhoA) are also located at the PM by its C-terminal isoprenylation. At the PM PaTox glycosyltransferase modifies a conserved tyrosine residue within the switch I (swI) region (in case of RhoA Y34). The DXD motif in PaTox is a typical motif for GT-A type of glycosyltransferases and essential to coordinate a divalent metal ion and in turn the sugar donor UDP-GlcNAc.
Typhimurium effector protein of the type III secretion system SPI-2 (Bhaskaran and Stebbins, 2012) and PMT, a toxin from Pasteurella multocida, which was recognized to activate heterotrimeric G proteins by deamidation (Kitadokoro et al., 2007; Orth et al., 2009; Orth and Aktories, 2012). Similar as PMT, the deamidase domain of PaTox activates the a-subunits of the heterotrimeric Gai and Gaq proteins by deamidation of Gln204 and Gln209, respectively (Orth et al., 2013). Moreover, PaTox induced activation of Gaq causes activation of Rho proteins (e.g. RhoA), thereby the preferred substrate of the PaTox glycosyltransferase is generated. Thus, it has been suggested that a functional interplay between the deamidase and glycosyltransferase domains of PaTox exists. This view is supported by the findings that the membrane guiding caused by the Nterminal helix of the PaTox glycosyltransferase is also essential for the activation of the deamidase domain of PaTox on heterotrimeric G-proteins (Jank et al., 2015).
toxins and effectors employed by bacteria in host pathogen interaction. In fact, our laboratory has indications that also tyrosine glycosylation is not unique for PaTox and is utilized as mechanism to modify host target proteins of other hosts than insects. Ethical statement We declare that there are no ethical conflicts, because this is a review article. Acknowledgments Studies performed in the laboratory of the authors were financially supported by the Deutsche Forschungsgemeinschaft (DFG, project Ak6 22/2). Transparency document
9. Conclusion Rho GTPases are well established protein substrates of various bacterial protein toxins and effectors acting on mammalian cells. Recent studies indicate that Rho protein are similarly important substrates for the action of bacterial toxin in insects. Therefore, the same principles of toxin activity, molecular mechanisms and structural requirements for toxin-substrate interactions appear to apply also for these hostepathogen interactions. Nevertheless, significant differences in toxin targeting have been reported. For example, up to now the modification of tyrosine residues by glycosylation is unique for PaTox. However, one has to consider that we only know the tip of the iceberg of the huge armamentarium of
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2015.05.017. References Adamson, P., Marshall, C.J., Hall, A., Tilbrook, P.A., 1992. Post-translational modifications of p21rho proteins. J. Biol. Chem. 267, 20033e20038. Aktories, K., 2011. Bacterial protein toxins that modify host regulatory GTPases. Nat. Rev. Microbiol. 9, 487e498. € sener, S., Just, I., Hall, A., 1989. The rho gene product Aktories, K., Braun, U., Ro expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3. Biochem. Biophys. Res. Commun. 158, 209e213. Aktories, K., Schwan, C., Papatheodorou, P., Lang, A.E., 2012. Bidirectional attack on the actin cytoskeleton. Bacterial protein toxins causing polymerization or depolymerization of actin. Toxicon 60, 572e581.
Please cite this article in press as: Jank, T., et al., Rho-modifying bacterial protein toxins from Photorhabdus species, Toxicon (2015), http:// dx.doi.org/10.1016/j.toxicon.2015.05.017
6
T. Jank et al. / Toxicon xxx (2015) 1e6
Bhaskaran, S.S., Stebbins, C.E., 2012. Structure of the catalytic domain of the Salmonella virulence factor SseI. Acta Crystallogr. D. Biol. Crystallogr. 68, 1613e1621. Bowen, D., Rocheleau, T.A., Blackburn, M., Andreev, O., Golubeva, E., Bhartia, R., ffrench-Constant, R.H., 1998. Insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280, 2129e2132. Bustelo, X.R., Sauzeau, V., Berenjeno, I.M., 2007. GTP-binding proteins of the Rho/ Rac family: regulation, effectors and functions in vivo. Bioessays 29, 356e370. Ciche, T., 2007. The biology and genome of Heterorhabditis bacteriophora. WormBook 1e9. Daborn, P.J., Waterfield, N., Silva, C.P., Au, C.P.Y., Sharma, S., ffrench-Constant, R.H., 2002. A single photorhabdus gene, makes caterpillars floppy (mcf), allows Escherichia coli to persist within and kill insects. Proc. Natl. Acad. Sci. U. S. A. 99, 10742e10747. Domenighini, M., Magagnoli, C., Pizza, M., Rappuoli, R., 1994. Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins. Mol. Microbiol. 14, 41e50. ment, R., Buisson-Touati, C., Flatau, G., Doye, A., Mettouchi, A., Bossis, G., Cle Gagnoux, L., Piechaczyk, M., Boquet, P., Lemichez, E., 2002. CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. Cell 111, 553e564. ffrench-Constant, R., Waterfield, N., 2006. An ABC guide to the bacterial toxin complexes. Adv. Appl. Microbiol. 58, 169e183. ffrench-Constant, R.H., Bowen, D.J., 2000. Novel insecticidal toxins from nematodesymbiotic bacteria. Cell. Mol. Life Sci. 57, 828e833. ffrench-Constant, R.H., Dowling, A., Waterfield, N.R., 2007. Insecticidal toxins from Photorhabdus bacteria and their potential use in agriculture. Toxicon 49, 436e451. Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Fiorentini, C., Boquet, P., 1997. Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine. Nature 387, 729e733. Forst, S., Nealson, K., 1996. Molecular biology of the symbiotic-pathogenic bacteria Xenorhabdus spp. and Photorhabdus spp. Microbiol. Rev. 60, 21e43. Gatsogiannis, C., Lang, A.E., Meusch, D., Pfaumann, V., Hofnagel, O., Benz, R., Aktories, K., Raunser, S., 2013. A syringe-like injection mechanism in Photorhabdus luminescens toxins. Nature 495, 520e523. Gerrard, J., Waterfield, N., Vohra, R., ffrench-Constant, R., 2004. Human infection with Photorhabdus asymbiotica: an emerging bacterial pathogen. Microbes. Infect. 6, 229e237. Gerrard, J.G., Joyce, S.A., Clarke, D.J., ffrench-Constant, R.H., Nimmo, G.R., Looke, D.F., Feil, E.J., Pearce, L., Waterfield, N.R., 2006. Nematode symbiont for Photorhabdus asymbiotica. Emerg. Infect. Dis. 12, 1562e1564. Heermann, R., Fuchs, T.M., 2008. Comparative analysis of the Photorhabdus luminescens and the Yersinia enterocolitica genomes: uncovering candidate genes involved in insect pathogenicity. BMC Genomics 9, 40. Hoffmann, C., Schmidt, G., 2004. CNF and DNT. Rev. Physiol. Biochem. Pharmacol. 152, 49e63. Hottiger, M.O., Hassa, P.O., Luscher, B., Schuler, H., Koch-Nolte, F., 2010. Toward a unified nomenclature for mammalian ADP-ribosyltransferases. Trends Biochem. Sci. 35, 208e219. Ishizaki, T., Naito, M., Fujisawa, K., Maekawa, M., Watanabe, N., Saito, Y., Narumiya, S., 1997. p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions. FEBS Lett. 404, 118e124. Jaffe, A.B., Hall, A., 2005. Rho GTPases: biochemistry and biology. Annu. Rev. Cell. Dev. Biol. 21, 247e269. Jank, T., Bogdanovic, X., Wirth, C., Haaf, E., Spoerner, M., Bohmer, K.E., Steinemann, M., Orth, J.H., Kalbitzer, H.R., Warscheid, B., Hunte, C., Aktories, K., 2013. A bacterial toxin catalyzing tyrosine glycosylation of Rho and deamidation of Gq and Gi proteins. Nat. Struct. Mol. Biol. 20, 1273e1280. Jank, T., Trillhaase, C., Brozda, N., Steinemann, M., Schwan, C., Suss, R., Aktories, K., 2015. Intracellular plasma membrane guidance of Photorhabdus asymbiotica toxin is crucial for cell toxicity. FASEB J. pii: fj.14e269381. [Epub ahead of print]. Just, I., Selzer, J., Wilm, M., Von Eichel-Streiber, C., Mann, M., Aktories, K., 1995a. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375, 500e503. Just, I., Wilm, M., Selzer, J., Rex, G., Von Eichel-Streiber, C., Mann, M., Aktories, K., 1995b. The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins. J. Biol. Chem. 270, 13932e13936. Kitadokoro, K., Kamitani, S., Miyazawa, M., Hanajima-Ozawa, M., Fukui, A., Miyake, M., Horiguchi, Y., 2007. Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin. Proc. Natl. Acad. Sci. U. S. A. 104, 5139e5144. Lang, A.E., Schmidt, G., Schlosser, A., Hey, T.D., Larrinua, I.M., Sheets, J.J., Mannherz, H.G., Aktories, K., 2010. Photorhabdus luminescens toxins ADPribosylate actin and RhoA to force actin clustering. Science 327, 1139e1142. Lemichez, E., Aktories, K., 2013. Hijacking of Rho GTPases during bacterial infection. Exp. Cell Res. 319, 2329e2336. Lerm, M., Pop, M., Fritz, G., Aktories, K., Schmidt, G., 2002. Proteasomal degradation of cytotoxic necrotizing factor 1-activated Rac. Infect. Immun. 70, 4053e4058.
Leung, T., Chen, X.Q., Manser, E., Lim, L., 1996. The p160 RhoA-binding kinase ROKa is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol. Cell. Biol. 16, 5313e5327. Madaule, P., Axel, R., 1985. A novel ras-related gene family. Cell 41, 31e40. Matsui, T., Amano, M., Yamamoto, T., Chihara, K., Nakafuku, M., Ito, M., Nakano, T., Okawa, K., Iwamatsu, A., Kaibuchi, K., 1996. Rho-associated kinase, a novel serine threonine kinase, as a putative target for the small GTP binding protein Rho. EMBO J. 15, 2208e2216. Meusch, D., Gatsogiannis, C., Efremov, R.G., Lang, A.E., Hofnagel, O., Vetter, I.R., Aktories, K., Raunser, S., 2014. Mechanism of Tc toxin action revealed in molecular detail. Nature 508, 61e65. Mohr, C., Just, I., Hall, A., Aktories, K., 1990. Morphological alterations of Xenopus oocytes induced by valine- 14 p21rho depend on isoprenylation and are inhibited by Clostridium botulinum C3 ADP-ribosyltransferase. FEBS Lett. 275, 168e172. Nobes, C.D., Hall, A., 1995. Rho, rac and cdc42 GTPases: regulators of actin structures, cell adhesion and motility. Biochem. Soc. Trans. 23, 456e459. Orth, J.H., Aktories, K., 2012. Molecular biology of Pasteurella multocida toxin. Curr. Top. Microbiol. Immunol. 361, 73e92. Orth, J.H., Fester, I., Siegert, P., Weise, M., Lanner, U., Kamitani, S., Tachibana, T., Wilson, B.A., Schlosser, A., Horiguchi, Y., Aktories, K., 2013. Substrate specificity of Pasteurella multocida toxin for alpha subunits of heterotrimeric G proteins. FASEB J. 27, 832e842. Orth, J.H., Preuss, I., Fester, I., Schlosser, A., Wilson, B.A., Aktories, K., 2009. Pasteurella multocida toxin activation of heterotrimeric G proteins by deamidation. Proc. Natl. Acad. Sci. U. S. A. 106, 7179e7184. Peel, M.M., Alfredson, D.A., Gerrard, J.G., Davis, J.M., Robson, J.M., McDougall, R.J., Scullie, B.L., Akhurst, R.J., 1999. Isolation, identification, and molecular characterization of strains of Photorhabdus luminescens from infected humans in Australia. J. Clin. Microbiol. 37, 3647e3653. Pfaumann, V., Lang, A.E., Schwan, C., Schmidt, G., Aktories, K., 2015. The actin and Rho-modifying toxins PTC3 and PTC5 of Photorhabdus luminescens: enzyme characterization and induction of MAL/SRF-dependent transcription. Cell. Microbiol. 17, 579e594. Popoff, M.R., 2014. Bacterial factors exploit eukaryotic Rho GTPase signaling cascades to promote invasion and proliferation within their host. Small GTPases 5, e28209. Popoff, M.R., Chaves-Olarte, E., Lemichez, E., Von Eichel-Streiber, C., Thelestam, M., Chardin, P., Cussac, D., Antonny, B., Chavrier, P., Flatau, G., Giry, M., de, G.J., Boquet, P., 1996. Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation. J. Biol. Chem. 271, 10217e10224. Ridley, A.J., Hall, A., 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70, 389e399. Ridley, A.J., Paterson, H.F., Johnston, C.L., Diekmann, D., Hall, A., 1992. The small GTPbinding protein rac regulates growth factor- induced membrane ruffling. Cell 70, 401e410. Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M., Aktories, K., 1997. Gln63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor 1. Nature 387, 725e729. Sehr, P., Joseph, G., Genth, H., Just, I., Pick, E., Aktories, K., 1998. Glucosylation and ADP-ribosylation of Rho proteins e effects on nucleotide binding, GTPase activity, and effector-coupling. Biochemistry 37, 5296e5304. Selzer, J., Hofmann, F., Rex, G., Wilm, M., Mann, M., Just, I., Aktories, K., 1996. Clostridium novyi a-toxin-catalyzed incorporation of GlcNAc into Rho subfamily proteins. J. Biol. Chem. 271, 25173e25177. Shao, F., Vacratsis, P.O., Bao, Z., Bowers, K.E., Fierke, C.A., Dixon, J.E., 2003. Biochemical characterization of the Yersinia YopT protease: cleavage site and recognition elements in Rho GTPases. Proc. Natl. Acad. Sci. U. S. A. 100, 904e909. Sheets, J.J., Hey, T.D., Fencil, K.J., Burton, S.L., Ni, W., Lang, A.E., Benz, R., Aktories, K., 2011. Insecticidal toxin complex proteins from Xenorhabdus nematophilus: structure and pore formation. J. Biol. Chem. 286, 22742e22749. Waterfield, N.R., Bowen, D.J., Fetherston, J.D., Perry, R.D., ffrench-Constant, R.H., 2001. The tc genes of Photorhabdus: a growing family. Trends Microbiol. 9, 185e191. Waterfield, N.R., Ciche, T., Clarke, D., 2009. Photorhabdus and a host of hosts. Annu. Rev. Microbiol. 63, 557e574. Waterfield, N.R., Wren, B.W., ffrench-Constant, R.H., 2004. Invertebrates as a source of emerging human pathogens. Nat. Rev. Microbiol. 2, 833e841. Wennerberg, K., Rossman, K.L., Der, C.J., 2005. The Ras superfamily at a glance. J. Cell. Sci. 118, 843e846. Wittinghofer, A., Vetter, I.R., 2011. Structure-function relationships of the G domain, a canonical switch motif. Annu. Rev. Biochem. 80, 943e971. Worby, C.A., Mattoo, S., Kruger, R.P., Corbeil, L.B., Koller, A., Mendez, J.C., Zekarias, B., Lazar, C., Dixon, J.E., 2009. The fic domain: regulation of cell signaling by adenylylation. Mol. Cell. 34, 93e103. Yarbrough, M.L., Li, Y., Kinch, L.N., Grishin, N.V., Ball, H.L., Orth, K., 2009. AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling. Science 323, 269e272.
Please cite this article in press as: Jank, T., et al., Rho-modifying bacterial protein toxins from Photorhabdus species, Toxicon (2015), http:// dx.doi.org/10.1016/j.toxicon.2015.05.017
