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1 Introduction

The expression of virulence during infection is intimately linked to the capacity of a bacterium to corrupt, either directly or indirectly, the actin cytoskeleton organization and dynamics. One of the most direct examples of this intimate crosstalk is represented by the C2 toxin from Clostridium botulinum, which ADP-ribosylates actin monomers, thereby promoting actin filament disassembly (Aktories et al. 1986). Following this seminal work, the last three decades have witnessed the discovery of an unprecedented convergence of virulence factors targeting not only actin monomers but also a subset of small Rho GTPases (Chardin et al. 1989; Paterson et al. 1990; Aktories 2015). These small Rho GTPases that belong to the superfamily of p21-Ras GTPases are critical upstream regulators of the actin cytoskeleton and several cell signaling pathways controlling gene expression. The targeting of Rho GTPases by this family of virulence factors confers major virulence traits upon microbial pathogens of humans, animals, and crops, thereby having a major impact on public health and the economy (Lemichez and Aktories 2013).

The pathogenic potential of bacteria is largely defined by their production of specific virulence factors targeting key host factors (Lemichez and Barbieri 2013). Their contribution to virulence, on the other hand, depends on the expression of host factors, such as adequate toxin receptors and cellular targets. For example, unlike humans, mice are resistant to diphtheria toxin due to a few variations in the amino acid sequence of the toxin receptor. Among these virulence factors, one can distinguish enzymes that affect the regulation of host factors that are crucial to the maintenance of cell homeostasis (Ribet and Cossart 2010). Examples of such virulence factors include the so-called bacterial AB toxins, such as the glucosylating toxins (TcdA and TcdB) from C. botulinum (Just et al. 1995) or the CNF1 toxin from pathogenic strains of Escherichia coli (Flatau et al. 1997; Schmidt et al. 1997). These toxins function as molecular syringes composed of an enzyme (the A-polypeptide) and one to several functional peptides (B-polypeptides) for host cell binding and the translocation of the A-domain into the cytosol. This mode of intoxication, which is independent of the presence of bacteria, is particularly interesting for studying the impact of intoxication on innate immune signaling in the absence of conserved bacterial components, such as lipopolysaccharide, and for developing tools in medicine. The other mode of injection of virulence enzymes that has been developed by bacteria consists of a direct injection of these enzymes by cell-bound bacteria through needle-like appendages or injectisomes, such as the type-III secretion system from Salmonella (Erhardt et al. 2010). Here, an assortment of different types of virulence effectors is injected into host cells (Galan 2009). These virulence effectors target several host proteins, acting cooperatively, synergistically, antagonistically or/and redundantly.

Studying the crosstalk between bacterial virulence factors and Rho GTPases continues to provide us with an invaluable source of information on the function and the regulation of Rho proteins, as well as their importance at the different stages of infection. Ultimately, these studies may provide us with a more comprehensive view on how evolutionary pressures might have operated to select such a remarkable diversity of virulence factors acting on small Rho GTPases and actin (Boquet and Lemichez 2003).

2 Switching On and Off the Rho GTPases with Virulence Factors

2.1 General Aspects of Rho GTPases

The small GTPases of the Rho family are essential regulatory switches that belong to the p21-Ras superfamily of GTPases (Heasman and Ridley 2008). Primarily, Rho proteins fulfill the critical function of controlling the architecture and dynamics of the actin cytoskeleton via their association with multiple effectors (Heasman and Ridley 2008). RhoA, Rac1, and Cdc42, the most studied Rho proteins, were discovered for their capacity to promote the formation of actin stress fibers, membrane ruffles and filopodia, respectively (Ridley and Hall 1992; Ridley et al. 1992; Nobes and Hall 1995) (Fig. 1). These Rho proteins are encoded by essential genes (Heasman and Ridley 2008). These GTPases also interfere with the signaling pathways controlling gene expression, notably those involved in the regulation of NF-κB-driven inflammatory and apoptotic processes (Jaffe and Hall 2005). The switch of Rho GTPases between an active and inactive form is under stringent spatiotemporal control via multiple interactions with a wide range of regulators. These regulators can be divided into three main categories (Fig. 2). Briefly, the G-domains of small GTPases form a canonical motif of highly conserved amino acids involved in the binding and hydrolysis of guanosine triphosphate (GTP) into guanosine diphosphate (GDP) (Wittinghofer and Vetter 2011). The guanine nucleotide-based conformational switch of these proteins involves the hydrolysis of GTP that is stimulated by GTPase-activating proteins (GAPs) (Moon and Zheng 2003) and GDP/GTP exchange by guanine nucleotide exchange factors (GEFs) (Rossman et al. 2005). The hydrolysis of the gamma-phosphate in GTP is catalyzed by a conserved glutamine residue in the switch-II region of small GTPases (Q61 for Rac1/Cdc42 and 63 for RhoA) that is strictly conserved among canonical small GTPases (Wittinghofer and Vetter 2011). In addition, contacts between the gamma-phosphate of GTP and threonine-35 in Rac1 (37 in RhoA) stabilize the switch-I region, which adopts a conformation prone to bind to CRIB-like domain-containing effectors for activation (Burbelo et al. 1995; Manser et al. 1998). In contrast to these highly conserved switch regions, the divergent carboxy-terminal portions of small GTPases contribute to confer specificity of interactions with effectors by promoting additional interactions and promote the interaction of the small GTPases with negatively charged lipids (Yeung et al. 2006; Lam and Hordijk 2013). Finally, small Rho GTPases are prenylated at their carboxy-termini. Therefore, Rho proteins remain cytosolic due to their interaction with guanine dissociation inhibitors (GDIs), which mask the hydrophobic prenyl group (DerMardirossian and Bokoch 2005; Navarro-Lérida et al. 2012). GDI also protects the Rho proteins from being targeted to proteasomal degradation (Boulter et al. 2010). Rac1 is palmitoylated after its geranylation for partitioning into membrane micro-domains, where it controls actin reorganization (Navarro-Lérida et al. 2012) (Fig. 2). Small Rho GTPases are thus switch proteins that oscillate between the cytosolic GDP-bound “off” form and a GTP-bound active form that is engaged in a series of intermolecular interactions at specific membrane locations to transduce specific signals.

Fig. 1
figure 1

Rho-, Rac-, and Cdc42-mediated actin cytoskeleton organization. Typical F-actin structures observed in different cell types treated with CNF1 toxin. From left to right: Vero cells displaying stress fibers (RhoA-like phenotype), HEp-2 cells displaying membrane ruffles (Rac1-like phenotype) and HEK293 cells displaying filopodial extensions (Cdc42-like phenotype). The cells were treated with 10−9M CNF1 for 24 h, and the actin cytoskeleton was labeled with FITC-modified phalloidin. Scale bar, 5 μm

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Fig. 2
figure 2

GTPase-based spatiotemporal cycle. Rho proteins orchestrate the timely local organization of complex arrays of protein-protein interactions. This involves the binding and hydrolysis of guanosine 5′-triphosphate (GTP) into guanosine 5′-diphosphate (GDP). Several residues, including glutamine-61 of Rac1 (Q63 in RhoA), are essential for the hydrolysis of the gamma-phosphate of GTP. Transitions between the guanine nucleotide-bound forms of the Rho proteins produce conformational changes in two flexible regions referred to as the switch-I and II regions. Binding to GTP enables the switch-I region to bind to and activate downstream effector proteins. The transition between the GTP/GDP forms of Rho is catalyzed by a large number of proteins containing GEF (guanine nucleotide exchange factors) or GAP (GTPase-activating protein) domains. The targeting of Rho proteins to cellular membranes for their activation is regulated by guanine nucleotide dissociation inhibitor (GDI) factors that retain the GDP-bound form in the cytosol. Following geranylation, Rac1 can incorporate palmitate at cysteine-178, which likely stabilizes its GTP-bound form and its association with detergent-resistant (liquid ordered) membrane domains (DRMs) (Navarro-Lérida et al. 2012)

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2.2 Bacterial Factors Targeting Rho GTPases

There is an astonishing convergence of virulence factors toward small Rho GTPases (Aktories 2011, 2015) (see examples in Fig. 3). These factors primarily target RhoA and Rac GTPases (Aktories 2011, 2015). Strikingly, this convergence on a group of proteins of small surface contrasts with the high degree of diversity of structures, activities, and modes of action of these virulence factors (Aktories 2011, 2015). We distinguished factors that mimic regulators of Rho proteins, i.e., GEFs, GAPs, and GDIs, from those that catalyze irreversible posttranslational modifications (Galan 2009; Aktories 2011, 2015). In terms of irreversible posttranslational modifications, we distinguished the factors that target the switch-I region of Rho from those targeting the switch-II (Fig. 3). These two different types of posttranslational modifications of switch domains inhibit or activate Rho proteins, respectively. All known factors that target the switch-II domain of Rho modify a glutamine residue (61 in Rac1 or 63 in RhoA), which catalyzes the hydrolysis of the gamma-phosphate (Wittinghofer and Vetter 2011). The different types of toxin or virulence factors-mediated posttranslational modifications targeting this glutamine residue encompass deamidation, transglutamination and ADP-ribosylation (Aktories 2011). This is of broad interest considering that Rho GTPases are also controlled by posttranslational modifications in cells (Visvikis et al. 2010). Posttranslational modifications or direct mutations of this key glutamine residue provoke a permanent activation state of the GTPases. In contrast, when several amino acids of the switch-I effector-binding region are modified, the GTPase signaling capacities are interrupted by blocking the interactions with effector proteins. These residues include RhoA threonine 37 (35 in Rac1/Cdc42), tyrosine 34 (32 in Rac1 and Cdc42) or asparagine 41 in RhoA (39 in Rac1/Cdc42) (Aktories 2015) (Fig. 3). A variety of posttranslational modifications affect the function of these amino acids, comprising ADP-ribosylation, several types of glycosylations, adenylylation, and deamidation (Aktories 2015; Aubert et al. 2016). In addition, the carboxy-terminal portion of Rho proteins can be proteolyzed by YopT from Yersinia spp. and LopT from Photorhabdus luminescens (Aktories 2011; Shao et al. 2003). In conclusion, these families of virulence factors are extremely diverse in terms of structures, enzymatic activities, and target different amino acids residues on Rho proteins for activation or inactivation. Recent studies on the glucosyltransferase domain of the lethal toxin (TcsL) of Clostridium sordellii revealed that efficient modification of small GTPases occurs at the interface with membranes, for example enriched in phosphatidylserine (Mesmin et al. 2004). Deciphering how toxic reactions occur at the interface with membranes is likely key to better understand many aspects of toxin specificity and mode of action.

Fig. 3
figure 3

Convergence of posttranslational modifications toward Rho GTPases. The target represents the structure of the GTPase RhoA. This diagram shows key amino acids (yellow) that are targeted by bacterial virulence factors. Examples of pathogens targeting Rho GTPases and the different types of biochemical modifications at each amino acid position are indicated. For exhaustive information see (Aktories 2011)

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2.3 Targeting Rho GTPases in Infection

It is still unclear when and how this remarkable diversity of factors corrupting Rho GTPases has been selected to benefit the bacteria. Indeed, they are produced by a large diversity of bacteria that are pathogenic in hosts from insects to mammals. This unprecedented convergence of virulence factors toward such small host proteins probably reflects the remarkable primary sequence conservation among these GTPases in all eukaryotes. The selective pressure may be related to the critical functions of Rho GTPases, notably during phagocytosis, which is conserved in amoebas that are natural predators of bacteria in the soil (Vlahou et al. 2009). A striking example of potential positive selective pressure is provided by the TccC3 toxin component from P. luminescens, which ADP-ribosylates the Rho GTPases (Lang et al. 2010). These toxin-producing bacteria can colonize the gastrointestinal tract of infective entomopathogenic nematodes to serve a symbiotic function. P. luminescens are released by the worm once it reaches the gastrointestinal tract of insect larvae, thereby offering a way to compromise the insect immune system and promote the development of worms in the insects. TccC3 is also toxic for human cells and thus represents a potential danger if transferred horizontally to human pathogens (Lang et al. 2010).

3 Ubiquitin and Proteasomal System Control of Rho GTPases

3.1 Ubiquitin and Proteasomal Machineries

Degradation of cellular proteins by the ubiquitin and proteasomal system (UPS) is implicated in a large number of biological functions, comprising signaling and immunity, and is deregulated in a large number of human diseases (Ashida et al. 2014). Ubiquitin is a protein of small size (8.5 kDa) that is ubiquitously expressed in tissues of eukaryotes. Ubiquitin can be cross-linked to lysine residues on the target after a series of transfer reactions between ubiquitin carrier proteins (Swatek and Komander 2016). Among these factors, the E3 ubiquitin-ligases (E3L) confer the specificity to the reaction by binding specifically to target proteins. Of note, the degradation of host cell factors can also be triggered by bacterial E3L-like toxins completing the arsenal of pathogens (Ashida et al. 2014). Additional molecules of ubiquitin can be subsequently attached to one of the seven lysines of the previously cross-linked ubiquitin molecule, leading to the formation of various types of poly-ubiquitin chains, such as Lysine-48 (K48) poly-ubiquitylation for substrate targeting to proteasomal destruction (Swatek and Komander 2016; Grabbe et al. 2011). Posttranslational modifications of proteins by mono-, multi-, and poly-ubiquitylation form a repertoire of modifications, allowing specific interactions with multiple families of ubiquitin-binding domain-containing proteins (Swatek and Komander 2016; Grabbe et al. 2011). If not degradative, protein ubiquitylation can be reverted by the action of de-ubiquitylating enzymes (Reyes-Turcu et al. 2009). Ubiquitylation is now viewed as a system of molecular barcodes controlling protein sorting at the membrane, as well as, local and/or temporal activation of proteins and specific inhibition by degradation.

3.2 Control of Rho GTPases by Ubiquitin and Proteasomal Machineries

Targeted proteolysis of proteins is likely the most efficient way to halt or attenuate the transduction of signals, and it has been exploited by potent toxins to corrupt host cells, such as the lethal factor from Bacillus anthracis, which cleaves MAPK kinases; AIP56 from Photobacterium damselae piscicida, which cleaves NF-κB; or YopT from Yersinia spp., which cleaves the carboxy-terminal portion of Rho proteins (Duesbery et al. 1998; Silva et al. 2013; Shao et al. 2003). The cellular ubiquitylation machinery is a direct target of bacterial virulence factors also restraining the activation of Rho GTPases triggered by several bacterial toxins (Doye et al. 2002; Visvikis et al. 2010) (Table 1). For example, once they are injected into the host cell cytosol, CHBP from Burkholderia pseudomaleimi and Cif from enteropathogenic E. coli preferentially catalyze the selective deamidation of the glutamine residue Q40 in ubiquitin and in Nedd8 ubiquitin-like molecule (Cui et al. 2010). The crosslinking of Nedd8 to the Cullin subunits of Cullin-RING E3 ubiquitin-ligases (CRLs) is critical for their activation (Cui et al. 2010). Therefore, the posttranslational modification of Nedd8 has a blocking effect on CRLs, in particular the Cullin3/Roc1/BACURD complex, which controls the level of total RhoA and therefore its activity, thereby promoting the formation of actin cables (Chen et al. 2009; Jubelin et al. 2010). In addition, the local activation of RhoA and the duration of its signaling activity is controlled by the ubiquitin-mediated degradation of the GTP-bound form of RhoA, which is catalyzed by the HECT-domain containing E3L Smurf1 (Wang et al. 2003; Ozdamar et al. 2005; Boyer et al. 2006b). The Cytotoxic Necrotizing Factor-1 (CNF1) from E. coli provides us with a second example of interference of UPS with bacterial virulence. Here, the study of CNF1 has been instrumental in unraveling the control of Rho GTPases by UPS and in unveiling the E3L targeting Rac1 (Doye et al. 2002; Visvikis et al. 2010). CNF1 belongs to a group of dermonecrotic toxins and virulence factors injected by the Type-3 secretion system (TSS3) found in E. coli, Yersinia pseudotuberculosis, Bordetella, and Vibrio. CNF1 toxin catalyzes the deamidation of the glutamine-61 residue in Rac1 or Cdc42 and the equivalent glutamine-63 in RhoA, thereby inhibiting their intrinsic and GAP-stimulated GTPase activity (Lerm et al. 1999; Flatau et al. 1997; Schmidt et al. 1997). Once Rac1 is activated, it is sensitized to ubiquitin-mediated proteasomal degradation (Doye et al. 2002). This downregulation of Rho signaling by UPS was initially recognized as a means for cells to control the duration and extent of G-protein signaling. In support of this hypothesis is the finding that activation of Rac1 by the GEF-domain of Dbl sensitizes this GTPase to ubiquitylation. Again, the activity of CNF1 was essential to screen for the E3 ubiquitin-ligase responsible for the degradation of Rac1 (Torrino et al. 2011). The regulation of Rac1 by HACE1 was confirmed by independent groups, which extended these findings to show the importance of this regulation in the control of cell migration and reactive oxygen species production (Castillo-Lluva et al. 2013; Goka and Lippman 2015; Daugaard et al. 2013). The activated form of Rac1 binds to the HECT-domain and ankyrin-repeat containing E3 ubiquitin protein ligase 1 (HACE1) and is prone to poly-ubiquitylation by HACE1 E3 ubiquitin-ligase activity (Torrino et al. 2011). In vitro experiments have established the high level of specificity of ubiquitylation of the GTP-bound form of Rac1 (Torrino et al. 2011). Depletion of HACE1 leads to an increase in the Rac1-dependent migration of mouse embryonic fibroblasts and increase of NADPH-mediated production of ROS (Castillo-Lluva et al. 2013; Daugaard et al. 2013). The importance of the regulation of UPS in the control of Rho GTPases continues to be unveiled with recent findings implicating the involvement of Nedd4L E3L in the proteasomal degradation of RhoB (Zhang et al. 2016).

Table 1 Rho GTPase ubiquitylation machinery
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4 Corruption of the Endothelial Barrier

4.1 Induction of Cellular Dewetting by Toxins

The endothelium forms a critical semipermeable barrier that prevents the dissemination of bacteria once they reach the bloodstream (Lemichez et al. 2010). The actin-myosin cytoskeleton and its upstream regulators largely control the barrier function of the endothelium (Bazzoni and Dejana 2004). RhoA signaling controls non-muscle myosin-II (NMII)-dependent F-actin fasciculation into stress fibers and contractility. In addition, RhoA establishes a coherent actomyosin network that transmits forces across the cytoplasm to equilibrate centripetal forces exerted at the edges of cells (Cai et al. 2010). A series of recent studies established that the breaching of the coherent actomyosin network in endothelial cells via direct inactivation of RhoA or inhibition of the Rho kinase ROCK with Y-27632 promotes the spreading of cells and, as a consequence, triggers the opening of transendothelial cell macroaperture (TEM) tunnels (Boyer et al. 2006a; Lemichez et al. 2012) (Fig. 4). Opening and widening of TEMs occur via a newly identified process, referred to as cellular dewetting (Gonzalez-Rodriguez et al. 2012; Lemichez et al. 2012). They open following disruption of actin cytoskeleton cohesion, due for example to an increase of the flux of cyclic AMP and consequently the downstream activation of the PKA and the exchange protein directly activated by cAMP (Epac)/Rap pathways (Maddugoda et al. 2011). This phenomenon is exacerbated by bacterial adenylate-cyclase toxins from B. anthracis and Bordetella spp. (Maddugoda et al. 2011). TEMs open transiently. Major progress has been made in identifying the physical principles governing the widening of TEMs and the molecular machinery that drives their closure (Maddugoda et al. 2011; Gonzalez-Rodriguez et al. 2012). Indeed, the opening of TEMs is a newly described cellular form of liquid dewetting. This common phenomenon occurs when a viscous liquid is forced to spread on a hydrophobic substrate, similar to heated oil on a nonstick pan (de Gennes et al. 2004). With regard to liquid dewetting, the aperture enlarges up to the formation of droplets as TEMs reach a maximal size in cells. Therefore, the cellular dewetting theory allows us to postulate the existence of a linear tension at the edge of TEMs, which equilibrates the membrane tension that pulls on the edges (Gonzalez-Rodriguez et al. 2012). TEMs represent not only a challenging model of direct crossing of the endothelium by pathogenic bacteria and/or their toxins but also a remarkable model system to investigate the relationships between cell architecture and cytoskeletal organization. This idea is supported by the finding that the inverse-Bin, Amphiphysin and Rvs, or (I-BAR) domain-containing protein missing in metastasis (MIM) senses via its I-BAR domain the curved edge of TEMs, where it accumulates soon after the opening (Maddugoda et al. 2011; Saarikangas et al. 2009). The recruitment of MIM in part controls the linear tension, as well as the later recruitment and activation of Arp2/3 complex that promotes the extension of membrane waves that close the TEMs (Maddugoda et al. 2011; Gonzalez-Rodriguez et al. 2012).

Fig. 4
figure 4

Opening of transendothelial cell macroaperture (TEM) tunnels. a, b Series of video images of the opening (a) and closure (b) of a transendothelial cell macroaperture (TEM) tunnel in a HUVEC intoxicated with the EDIN homologue C3-exoenzyme from Clostridium botulinum 24 h. Bar = 10 µm. c Schematic representation of TEM tunnel opening that results from the spreading of cells due to a reduction of contractility of the actomyosin cytoskeleton that promotes an increase of membrane tension. Green and red arrows represent forces at play around the edge of transcellular tunnels together with the distance between membranes determined by atomic force microscopy

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4.2 Invasive Properties of Virulence Factors Targeting RhoA

Most bacterial toxins target various Rho GTPases, with the exception of the group of C3-like ADP-ribosyltransferases, which catalyze the ADP-ribosylation of RhoA (Wilde et al. 2003). C3-like exoenzymes consist of a single polypeptide chain that penetrates into the host cells via an undefined molecular mechanism. C3-like factors are isolated from different strains of Clostridia, as well as from Staphylococcus aureus, among which there are three isoforms, EDIN-A, EDIN-B and EDIN-C (Yamaguchi et al. 2001). ADP-ribosylation of RhoA promotes its tight association with RhoGDI, leading to the sequestration of an inactive form of the GTPase in the cytosol (Fujihara et al. 1997; Genth et al. 2003). Most recently, genetic evidence demonstrated the invasive properties conferred by edinB to S. aureus in a model of pneumonia-induced bacteremia (Courjon et al. 2015). Indeed, the deletion of edinB reduced the risk of bacteremia by 50 % in animals suffering from pneumonia triggered by the European lineage community-acquired methicillin resistant S. aureus (CA-MRAS) strain ST80-MRSA-IV. This property was restored upon the re-expression of wild type (WT) EDIN-B but not by the expression of a catalytically inactive mutant. The presence of edinB does not improve the capacity of S. aureus to persist in the bloodstream, reinforcing the idea that this factor acts by promoting bacterial translocation through the successive pulmonary alveolar epithelial and endothelial barriers rather than by promoting an anergy of phagocytes in the context of the bloodstream (Courjon et al. 2015; Munro et al. 2010). Of note, EDIN-like factors such as C3 from C. botulinum may interfere with immune cells in other context (Barth et al. 2015). This dual role of breaching the epithelial and endothelial barriers through the inhibition of RhoA is consistent with previous cell biology studies that had implicated RhoA in the control of tight junction stability by studying C3 from C. botulinum (Nusrat et al. 1995). All these findings are in accordance with a combination of epidemiological surveys that point to a critical role for EDIN factors in the dissemination of S. aureus and the induction of septic metastasis (Lemichez et al. 2012).

5 Corruption of Innate Immunity

5.1 Virulence Factors Activating Rho GTPases and the Inflammasome

We must address rather conflicting relationships with the bacterial world. On one hand, we have learned how to confine and tolerate bacteria of the gut flora, which contribute to essential functions in digestion and help maintain our body homeostasis (Sansonetti 2011; Brodsky and Medzhitov 2009). On the other hand, our bodies must discriminate bacteria that are pathogenic and therefore express distinct combinations of toxins that corrupt the host cells homeostasis (Brodsky and Medzhitov 2009; Stuart et al. 2013; Lemichez and Barbieri 2013). Several studies have recently revealed that toxins activating Rho GTPases found in extra-intestinal pathogenic strains of E. coli and in some strains of Yersinia promote acute inflammatory responses (Diabate et al. 2015; Munro et al. 2004; Schweer et al. 2013). The importance of Rho-activating factors such as SopE, SopE2 and SopB in Salmonella-induced inflammatory responses has also been demonstrated (Bruno et al. 2009). Acute inflammatory responses involve a combination of (i) a priming-phase of gene expression of inflammatory cytokines and chemokines, also comprising pro-inflammatory IL-1β/IL18 in immune cells, as well as (ii) the establishment of an execution-phase that involves the organization of the inflammasome complex for IL-1β/IL18 processing and secretion (Zhao and Shao 2016; Broz and Monack 2011). The induction of expression of inflammatory mediators triggered by Rho-activating factors, in absence of other bacterial factors, is consistent with initial studies that have determined the capacity of Rho GTPases, notably Rac1 and Cdc42, in promoting c-Jun, p38 MAP kinase and NF-κB signaling pathway activation (Minden et al. 1995; Coso et al. 1995; Perona et al. 1997; Munro et al. 2004). More recent studies have revealed the importance of Rho GTPase activity combined with that of Nucleotide-binding Oligomerization Domain (NOD) and the RIP-kinases components for efficient NF-κB-dependent transcription of inflammatory mediators in response to virulence factors and conserved bacterial components (Arbibe et al. 2000; Keestra et al. 2013; Boyer et al. 2011; Stuart et al. 2013; Kawano et al. 2010). Variations of hierarchy and/or interplay between these factors have been analyzed in different model organisms and contexts of infection (Stuart et al. 2013). Importantly, this capacity of cells to sense the imbalance of cell signaling triggered by virulence factors activating Rac1/Cdc42 GTPases has been proposed as a general means to discriminate between the presence of commensal bacteria versus pathogen attack (Stuart et al. 2013; Keestra et al. 2013; Xu et al. 2014). Paralleling their effects on gene expression, the activation of Rac1 and Cdc42 by virulence factors can directly engage inflammasome signaling to promote IL-1β secretion, which plays a major role in the execution of efficient programs of pathogen eradication (Stuart et al. 2013). For example, the inflammation of the gut epithelium that is elicited by Salmonella typhimurium infection is triggered by the activation of caspase-1 signaling, which is mediated by SopE at the level of enterocyte mainly (Muller et al. 2009). The SopE-mediated activation of caspase-1 in vitro is mediated by Rac1 and Cdc42. We have begun to better appreciate how some bacteria deal with these anti-bacterial responses, although this has to be better characterized mechanistically. In the context of the bacteremia induced by CNF1-producing extra-intestinal strains of E. coli, IL-1β-mediated host responses promote an efficient destruction of bacteria and favors host survival (Diabate et al. 2015). Therefore, in this context, the CNF1 toxin acts as an anti-virulence factor. These detrimental responses for bacteria are specifically blunted by the concurring action of the pore-forming toxin α-hemolysin from E. coli (Diabate et al. 2015). This α-hemolysin acts downstream of Rac1 activation to prevent IL-1β secretion (Diabate et al. 2015). In conclusion, the combined sensing of conserved products of bacteria and the specific virulence factor activity, notably toward Rho GTPases, synergistically modulate the level of inflammasome-mediated anti-microbial responses to efficiently fight pathogen attack. The precise consequences of the activation or inhibition of Rho GTPases remain to be fully deciphered, as does the importance of actin cytoskeleton remodeling in this context. Some pathogens respond to these host countermeasures by the production of a second set of factors that blunt virulence factor sensing.

5.2 RhoA-Inactivating Toxins and the Inflammasome

Normally in our body the bacterial flora is confined by the joint action of physical mucoepithelial barriers, the immune system and growth limitation promoted by inter-species competition (Sansonetti 2011; Brodsky and Medzhitov 2009). The induction of bacterial imbalance or dysbiosis triggers deadly inflammatory bowel diseases (DuPont and DuPont 2011). One such example is provided by Clostridium difficile, which can overgrow following antibiotic treatment and promote diseases ranging from diarrhea to pseudomembranous colitis, which is potentially fatal. Several strains of C. difficile produce two potent toxins (TcdA and TcdB), which glucosylate and inactivate RhoA, Rac1, and Cdc42 (Just et al. 1995). Cell biology studies have established the capacity of these toxins to disrupt the function of the epithelial barrier, establishing the importance of RhoA and Rac1 in the control of adherent and tight junctions (Boquet and Lemichez 2003; Lemichez and Aktories 2013). Following toxin action, the diffusion of bacterial conserved factors across the epithelial barrier likely contributes to stimulate the gut immune system. Recent new exciting findings highlighted how these large clostridial toxins targeting RhoA promote directly inflammatory responses. Indeed, the glucosyltransferase activity of TcdB in bone marrow-derived macrophages (BMDM) activates the nucleotide-binding oligomerization domain receptors (NLR) protein Pyrin, which in turn mediates the activation of the ASC/Caspase-1 inflammasome complex for interleukin-1 beta processing and secretion (Xu et al. 2014). Study of this mode of activation of pyrin and the inflammasome is of high importance considering that activating mutations in the pyrin-encoding gene MEFV also cause an auto-inflammatory disease known as Mediterranean fever (French 1997). Other NLR factors, including NOD1/2, Nlrp3, Nlrc4 and AIM2 are not involved. Toxins inhibiting small GTPases other than RhoA do not stimulate pyrin/inflammasome signaling (Xu et al. 2014). Activation of the inflammasome by RhoA inhibition results from the activity of several toxins targeting the switch-I domain at different positions and by different biochemical mechanisms (Xu et al. 2014). This holds true for the virulence factor TecA from Burkholderia cenocepacia that inhibits RhoA by deamidation of its asparagine 41 and an equivalent in Rac1 (Aubert et al. 2016). The toxic activity of TecA is perceived by host cells and converted into efficient activation of the pyrin inflammasome thereby restricting the infection and promoting animal survival (Xu et al. 2014; Aubert et al. 2016). This phenomenon of inflammasome activation does not involve the sensing of actin depolymerization as shown by cytochalasin-D treatment. Instead it appears to involve an uncharacterized indirect sensing of the consequences of RhoA inactivation (Xu et al. 2014).

6 Conclusions

In conclusion, during these last years, we have witnessed the discovery of an incredible variety of examples of virulence factors of pathogens targeting Rho GTPases. The coming years will probably see an enrichment of the repertoire of modifications of Rho and other targeted GTPases by newly described toxins, and the identification of host factors critical for posttranslational-based regulation of the activity of Rho GTPases, a so far less known area. Deregulations of the small Rho GTPases are implicated in a large variety of human diseases, comprising immunological disorders, mental retardations, and cancer. Much remains to be elucidated on this remarkable convergence on a few targets during infection. It is certainly important to determine in greater detail the action of factors targeting Rho GTPases at the different steps of infection. Ultimately, we might be able to use this knowledge to develop innovative therapeutic molecules and diagnostic tools in infectiology and vaccinology, as well as in degenerative diseases and oncology.