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Review

The Role of Rho GTPases in Toxicity of Clostridium difficile Toxins

by
Shuyi Chen
†,
Chunli Sun
†,
Haiying Wang
and
Jufang Wang
*
School of Bioscience and Bioengineering, South China University of Technology (SCUT), Guangzhou 510006, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Toxins 2015, 7(12), 5254-5267; https://doi.org/10.3390/toxins7124874
Submission received: 24 September 2015 / Revised: 18 November 2015 / Accepted: 18 November 2015 / Published: 2 December 2015
(This article belongs to the Collection Clostridium difficile/Clostridium sordellii and Clostridium perfringens Toxins)
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Abstract

:
Clostridium difficile (C. difficile) is the main cause of antibiotic-associated diarrhea prevailing in hospital settings. In the past decade, the morbidity and mortality of C. difficile infection (CDI) has increased significantly due to the emergence of hypervirulent strains. Toxin A (TcdA) and toxin B (TcdB), the two exotoxins of C. difficile, are the major virulence factors of CDI. The common mode of action of TcdA and TcdB is elicited by specific glucosylation of Rho-GTPase proteins in the host cytosol using UDP-glucose as a co-substrate, resulting in the inactivation of Rho proteins. Rho proteins are the key members in many biological processes and signaling pathways, inactivation of which leads to cytopathic and cytotoxic effects and immune responses of the host cells. It is supposed that Rho GTPases play an important role in the toxicity of C. difficile toxins. This review focuses on recent progresses in the understanding of functional consequences of Rho GTPases glucosylation induced by C. difficile toxins and the role of Rho GTPases in the toxicity of TcdA and TcdB.

Graphical Abstract

1. Introduction

Clostridium difficile (C. difficile), a strictly anaerobic, gram-positive and spore-forming bacillus, was identified as the major infectious cause in 1978 [1], leading to pseudomembranous colitis and antibiotic-associated diarrhea in human and animals [1,2,3]. Since 2000, C. difficile infections (CDIs) have been increasing in prevalence and becoming less responsive to treatment [4,5,6]. In the United States, the number of CDI hospital discharges has been more than doubled from 2001 (≈148,900 discharges) to 2005 (≈301,200 discharges) [4] and current estimates suggest that CDI patients are more than 500,000 annually with at least 14,000 deaths [7]. Moreover, the annual healthcare costs of patients with CDI have exceeded $1.5 billion in the United States [8], which would increase the financial burden of individuals and government.
C. difficile causes disease by the release of the two exotoxins, toxin A (TcdA) and toxin B (TcdB) [9,10]. TcdA is designated as an enterotoxin responsible for fluid accumulation in ileum, while TcdB is referred to be a cytotoxin with about 100- to 1000-fold higher cytotoxic potency than TcdA [11]. Both of these two toxins belong to the family of clostridial glycosylating toxins, which also includes Clostridium sordellii (C. sordellii) hemorrhagic toxin (TcsH) and lethal toxin (TcsL), and Clostridium novyi (C. novyi) α-toxin (TcnA) [10,11,12,13], causing gas gangrene syndromes [11]. The large clostridial glycosylating toxins target the families of Rho and Ras GTPases and modify them by mono-O-glucosylation (C. difficile TcdA and TcdB, C. sordellii lethal and hemorrhagic toxins) [14,15,16,17] or mono-O-N-acetylglucosaminylation (C. novyi α-toxin) [18], which inhibits the signaling and regulatory functions of these target proteins [13,14], resulting in host cell morphological changes [19], secretion inhibition [20], phospholipase D inactivation [21], apoptosis [22], phagocytosis disregulation [23] and other actin cytoskeleton and Rho GTPase dependent processes.
This review focuses on recent progresses in the understanding of functional consequences of Rho GTPases glucosylation induced by C. difficile toxins and the role of Rho GTPases in the toxicity of TcdA and TcdB.

2. Structure–Function Relationship and Mechanism of TcdA and TcdB

C. difficile TcdA and TcdB are both single-chain large protein toxins. TcdA consists of 2710 amino acid residues with molecular mass of 308 kDa and TcdB consists of 2366 residues with a mass of 270 kDa (Figure 1) [12,24]. It is believed that the toxins are comprised of multimodular structures, and on the basis of their amino acid sequences and tripartite structure [12,25,26], an ABCD model (Figure 1) was proposed recently for the structure–function relationship of the toxins [24]. The N-terminus harbors the biological active domain (A domain) with glucosyltransferase activity (glucosyltransferase domain, GTD) [27,28], and subsequently a cysteine protease domain responsible for autocleavage process (CPD, C domain) [29,30,31,32]. The receptor binding domain (RBD, B domain) is located at the C-terminus of the toxins and consists of combined repetitive oligopeptides (CROPs), which is considered to be involved in the receptor binding [25,33,34,35]. The crystal structure of the CROPs of TcdA has been revealed. The CROPs are composed of 31 short repeats and seven long repeats, with each repeat consisting of a β-hairpin followed by a loop. Furthermore, co-crystalization of TcdA with an artificial trisaccharide containing the Galα1-3Galβ1-4GlcNac-glycan confirms that carbohydrate binding occurs in the CROPs of TcdA [36,37]. The large region between the CPD and RBD is predicted to be the translocation domain (TD, D domain), which is critical for toxin delivery into the host cytosol via pore formation and membrane insertion [38,39,40,41,42,43].
Toxins 07 04874 g001 1024
Figure 1. The ABCD model of Clostridium difficile toxins (toxin B as example). A domain, containing the DXD motif, is located at the N terminus (GTD, red, amino acids 1–543) and harbors the glucosyltransferase activity. The C terminus is characterized as receptor binding domain (green, B domain), consisting of combined repeat oligopepides (CROPs). The cysteine protease domain (CPD, purple, amino acids 544–767) is involved in the auto-cleavage process of the toxins. The middle part of TcdB is the translocation domain (TD, gray, D domain), within which there is a short hydrophobic region (HR, oblique line, amino acids 956–1128). The TD is considered to be involved in pore formation, conformational changes and the delivery of the GTD and CPD.
Figure 1. The ABCD model of Clostridium difficile toxins (toxin B as example). A domain, containing the DXD motif, is located at the N terminus (GTD, red, amino acids 1–543) and harbors the glucosyltransferase activity. The C terminus is characterized as receptor binding domain (green, B domain), consisting of combined repeat oligopepides (CROPs). The cysteine protease domain (CPD, purple, amino acids 544–767) is involved in the auto-cleavage process of the toxins. The middle part of TcdB is the translocation domain (TD, gray, D domain), within which there is a short hydrophobic region (HR, oblique line, amino acids 956–1128). The TD is considered to be involved in pore formation, conformational changes and the delivery of the GTD and CPD.
Toxins 07 04874 g001
The molecular mode of action of the toxins is not completely understood, but there is a hypothetical model that is widely accepted (Figure 2). In this model, the toxins firstly bind to cell surface receptor(s) via the RBD, and then enter the host cells through endocytosis [44] to reach endosomal compartments. Under a low pH condition of endosome [45], conformational change [38,41] and pore formation [39,42] take place and eventually the GTD and CPD were delivered into the host cytosol [43], where inositol hexaphosphate (InsP6) activates the protease for auto-proteolytic cleavage and eventually the GTD is released [29,30,42] (Figure 2).
Toxins 07 04874 g002 1024
Figure 2. The molecular mode of action of Clostridium difficile toxins. The toxins bind to the cell surface receptors by the RBD (1) and the toxin-receptor complex is internalized (2); following endocytosis, the toxins reach the endosomal compartments (3); under acidic environment of the endosome, the toxins undergo conformational changes and pore formation (4); the CPD and GTD translocate across the endosomal membrane, and the auto-cleavage process takes place in the presence of hexakisphospate (InsP6) (5); and only the GTD of the N terminus of the toxins is released into the host cytosol, where the Rho proteins are glucosylated by the GTD (6).
Figure 2. The molecular mode of action of Clostridium difficile toxins. The toxins bind to the cell surface receptors by the RBD (1) and the toxin-receptor complex is internalized (2); following endocytosis, the toxins reach the endosomal compartments (3); under acidic environment of the endosome, the toxins undergo conformational changes and pore formation (4); the CPD and GTD translocate across the endosomal membrane, and the auto-cleavage process takes place in the presence of hexakisphospate (InsP6) (5); and only the GTD of the N terminus of the toxins is released into the host cytosol, where the Rho proteins are glucosylated by the GTD (6).
Toxins 07 04874 g002

3. Interaction of C. difficile Toxins and Rho GTPases

C. difficile toxins target Rho-GTPase proteins in the host cytosol. GTPases are molecular switches controlling many complicated cellular processes, which number over 60 in mammals and are divided into five major groups: Ras, Rho, Rab, Arf and Ran [46]. Rho GTPases are involved in numerous signaling processes, including the regulation of actin cytoskeletion, cell polarity, gene transcription, G1 cell cycle progression, microtubule dynamics, vesicular transport pathways, control of the activity of protein and lipid kinases, phospholipases, and nicotanimide adenine dinucleotide-oxidase [46,47,48]. Furthermore, in respect to host-pathogen interactions, Rho GTPases are essential for epithelial barrier functions, cell–cell contact, immune cell migration, phagocytosis and cytokine production [48].
Improvement in in vitro assays and mass spectrometry analysis from toxin-treated cells revealed broader spectrum and better detection in direct intracellular glucosylation of GTPases, respectively. Until now, the reported substrates of C. difficile toxins are mostly Rho subfamily proteins, including RhoA, B, C, Rac1–3, RhoG, Cdc42, TC10, but not RhoE nor RhoD [27,48]. Besides, TcdA was reported to modify Rap in vitro [49] and TcdB variant from C. difficile strain C34 is able to glucosylate R-Ras, Ral and Rap [50]. By contrast, C. sordellii lethal toxin can glucosylate Rac, Cdc42 and Ras proteins (Ras, Rap, Ral), but not (or much less) Rho [16]. Moreover, it was reported that the lethal toxin from C. sordelli strain IP82 is able to modify Rho, Rac, Ras and Ral, while lethal toxin from strain VPI9048 modifies Cdc42, Rac, Rho, Ras and Ral [51]. TcdA- and TcdB-induced glucosylation occurs at Thr37 of Rho and Thr35 of all other GTPases [15]. Thr35/Thr37, conserved in all low molecular mass GTPases, is located in the effector switch-I region of the GTPases and is essential for nucleotide binding and coordination of the divalent cation magnesium [52]. As a result, glucosylation of Thr35/Thr37 of the GTPases blocks the interaction of the GTPases with their effectors, such as GEFs, GAPs and GDIs [53,54].
The GTD covers the amino acid residues of 1–543 in TcdB and 1-542 in TcdA, respectively [55]. Based on the crystal structure of the GTD of TcdB [56], the Asp286-Xaa287-Asp288 (DXD) motif [27] and several key residues including Trp102, Asp270, Arg273, Tyr284, Asn384 and Trp520 [28] are revealed to be essential for Mn2+, UDP and glucose binding. C. difficile toxins use UDP-glucose as the cosubstrate, binding of which triggers the structure switch of the “flexible loop” of the GTD from an open, disordered conformation to a closed, ordered conformation [57] and creates a deep pocket that serves as a binding site for the acceptor substrate [58]. However, the substrate specificity of the GTP-binding proteins for the toxins has not been structurally defined to date, while distinct amino acids or regions on Rho GTPases have been shown to have a role in defining specificity [59,60].

4. The Role of Rho GTPase Glucosylation in Toxicity of TcdA and TcdB

Although the final consequence of Rho GTPase glucosylation is biological inactivation of the GTPases, a glucose moiety attached to the conserved threonine residue causes various alterations of Rho functions, including (1) inhibition of nucleotide exchange induced by GEFs; (2) inhibition of GTP hydrolysis stimulated by GAPs; (3) blocking of the Rho/guanine nucleotide dissociation inhibitors interaction; and (4) blocking of the coupling the Rho with effectors [11,13,14,53,54]. In addition to the GTPase cycling, the glucosylation of Rho GTPases also alters the cytosol-membrane cycling, in which the GDP-boud glucosylated Rho is entrap at the membranes and not able to form complex with GDI anymore [61]. As a result, the glucosylation completely blocks all Rho-dependent signaling pathways [11].

4.1. Cytopathic and Cytotoxic Effect

The Rho GTPases are the critical regulators of the actin cytoskeleton. It includes at least 20 members that can be subdivided into six groups: Rho subfamily (RhoA, RhoB, RhoC), Rac subfamily (Rac1, Rac2, Rac3, RhoG), CDC42 subfamily (CDC42, Wrch1, TC10, Chp, TCL), Rnd subfamily (Rnd1, Rnd2, Rnd3), Rho BTB subfamily (RhoBTB1, RhoBTB2, RhoBTB3) and Miro subfamily (Miro1, Miro2). Among them, Rho is responsible for the assembly of contractile actin and myosin filaments (stress fibers), while Rac and Cdc42 are involved in the formation of actin-rich surface protrusions (lamellipodia) and actin-rich, finger-like membrane extensions (filopodia), respectively [62,63]. The glucosylation modification caused by TcdA or TcdB mainly induces cytopathic effects that are characterized as loss of actin stress fibers, reorganization of cortical actin, disruption of intercellular junctions and increase in cell barrier permeability [11,64,65,66,67,68,69,70]. The cytopathic effects of the intoxicated cells are visualized as drastic morphological changes, such as shrinking and rounding of cells, and initially accompanied by formation of neurite-like retraction fibers [19]. Furthermore, differences in toxin’s substrate specificity lead to different cytopathic and cytotoxic effects [71]. For example, TcdA and TcdB from the strain VPI10463 cause morphological changes at fibroblasts with cell rounding and formation of “neurite-like” protrusions, and the intoxicated cells remain attach to the substratum. In comparison, variant TcdB from strains 1470 and 8864, as well as lethal toxin from C. sordellii, whose substrates are Rac1 and Ras-GTPases but not Rho, induce cell rounding with formation of filopodia-like structures, and moreover, with detachment of most intoxicated cells [71].
Although it is generally accepted that cytopathic effects are mainly caused by Rho GTPase inactivation, there are some controversies. Chen and coworkers found in 2002 that PKS signaling plays an important role in TcdA-mediated damage on tight junction structures and functions [72]; Furthermore, Kim and coworkers reported in 2009 that, when exposed to TcdA, the FAK and paxillin in human colonocytes were dephosphorylated by a direct interaction of TcdA with the catalytic domain of Src [73]. Nevertheless, it is still unclear which member(s) of Rho GTPases is/are responsible for cell rounding. At first, it was attributed to RhoA inactivation in TcdB intoxicated cells [15], however, Halabi-Cabezon and coworkers suggested later that Rac1, rather than RhoA or Cdc42, is crucial for the cytopathic effects induced by TcdA and TcdB [74].
Besides cytopathic effects, the C. difficile toxins can induce cytotoxic effects on the intoxicated cells. The intoxicated cells respond to RhoA inactivation with upregulation of the pro-apoptotic immediate early gene product RhoB, which transiently escapes glucosylation while being activated and is involved in the regulation of programmed cell death [75,76,77]. TcdA and TcdB are able to induce type I and type III programmed cell death. Type I programmed cell death, called apoptosis, is characterized as caspase activation, chromatin condensation and phosphatidylserin exposure, while type III programmed cell death, so called necrosis, is defined by ATP depletion, generation of reactive oxygen species, loss of membrane integrity and calpain/cathepsin activation [10]. However, the relationship between cytotoxic effects and Rho proteins glucosylation is still in debate up to this date. As Mahida Y.R. suggested, the cytotoxic effects induced by TcdA and TcdB are independent on glucosyltransferase activity, because of the direct targeting of toxins towards mitochondria [78]. On the contrary, several studies have suggested that, using glucosyltransferase-deficient mutant toxins or uridine 5′-diphosphate-2′,3′-dialdehyde to block the toxins’ enzymatic activity, the cytotoxic effects are dependent on TcdA and TcdB glucosyltranferase activity [79,80,81,82,83]. Thus, RhoA inhibition is responsible for apoptosis in endothelial cells [81,84]. Additionally, in respect to necrosis induced by C. difficile toxins, we found that structurally intact glucosyltransferase-deficient TcdA and TcdB are essentially devoid of glucosylation activity and cytotoxicity [85,86], while Chumbler and coworkers found that both wild-type TcdB and TcdB mutants with impaired autoprocessing or glucosyltransferase activities are able to induce rapid, necrotic cell death in HeLa and Caco-2 epithelial cell lines [87].

4.2. Immune Response

Although the role and contribution of C. difficile toxins to disease pathogenesis is being increasingly understood, the aspects of C. difficile-driven effects on host immunity remain rudimentary [88]. Following infection, both adaptive and innate arms of the host immune system are activated, leading to activation of the inflammasome and NFκB-mediated pathways [89]. Then, the NFκB-mediated pathways would lead to production of pro-inflammatory cytokines, which contribute to the initiation and propagation of inflammatory response. Both TcdA and TcdB can cause massive recruitment of neutrophils due to the stimulation of inflammatory mediators from colonocyte and immune cells [90]. Recently, using murine and human ex vivo infection models, Jafari and coworkers [88] found that, C. difficile modulates the host innate immunity via toxin-dependent and -independent mechanism, in which the majority of C. difficile-driven effects on murine bone-marrow-derived dendritic cell (BMDC) activation were toxin-independent, but the toxins were responsible for BMDC inflammasome activation. Besides, infected DC-T cell crosstalk revealed that the C. difficile strain 630 and R20291 were able to elicit a differential DC IL-2 family cytokine milieu, which culminated in significantly greater Th1 immunity in response to R20291. Thus, they suggested in a summary that C. difficile strains have evolved to actively modulate DC-T cell crosstalk and it is likely to be dictated by the genetic content of both the bacterium and the host [88].
As the main targets of many bacterial virulence factors, Rho proteins play an extremely important role in immune and defense functions of target cells against pathogens. Thus, the relationship between Rho GTPase glucosylation and host immune response has been studied. Xu and coworkers found that the glucosyltransferase-inactive mutant TcdB fails to induce inflammasome stimulation [91]. In contrary, Ng and colleagues suggested that the activation of inflammasome is independent on the catalytic function of TcdB, but depends on the recognition of intact toxin [92]. Furthermore, release of Rho-dependent or -independent cytokines induced by TcdA and TcdB has been observed [93,94,95], with NFκB or MAPK p38 functioning as critical molecules of cytokine secretion [93,96,97,98]. Recently, the role of C. difficile flagellin in the production of CXCL8/IL-8 and CCL-20 through the TLR5-dependent activation of NFκB and p38 MAP kinase pathways was addressed [99]. However, further studies are needed to determine the contribution of this response to the CDI pathogenesis. In 2014, we found immunization of BALB/C mice with TcdB-treated CT26 cells would elicited long-term, specific anti-tumor immunity response, and the effector function of the toxin’s glucosyltransferase activity seems to be necessary [100].
As a key signaling molecule and inflammation mediator, reactive oxygen species (ROS) were proposed to be stimulated by Rac proteins, which are required for NAD(P)H oxidase (NOX) activation in phagocytes [101,102] and nonphagocytes [103]. The observation that TcdB-induced glucosylation of Rac1 markedly diminished its ability to support the activity of superoxide-generating NOX in phagocytes [54] and nonphagocytes [104] was in line with this proposal, however, glucosylated Rac1 would not interfere with the process of NOX activation that unmodified Rac1 is involved [54]. Furthermore, robust production of ROS in TcdA or TcdB intoxicated cells or animals has been observed [92,105,106,107]. Recently, using siRNA transfection technology, Farrow and colleagues [106] found that TcdB-induced cell death in vitro depends on the assembly of NOX complex and the production of ROS in the host epithelial cells. They explained this apparent paradox as that the Rac-dependent NOX assembly occurs during the process of TcdB entry into endosomes, before the delivery of the TcdB GTD. Besides, they speculated that TcdB pulls their multiple receptors together with NOX complex in a unique way. Nevertheless, it needs further studies to support.

5. Conclusions and Future Considerations

C. difficile TcdA and TcdB are the major virulent factors of CDI. The molecular mode of action of the toxins is not completely understood currently, but it has been considered that the toxicity of the toxins depends on the glucosyltransferase activity of TcdA and TcdB. However, many studies suggest that some responses observed with TcdA and TcdB may not be simply explained by toxin-induced glucosylation of the Rho GTPases, of which controversial role may exist in other effects caused by the toxins. For instance, as reported in these decades, Rho inactivation blocks the NFκB pathway, the transcription and secretion of TLRs-induced inflammatory cytokines [108,109] and chemoattractants [110], all of which, interestingly, seem to be stimulated by C. difficile toxins [98,111]. Moreover, the p38 MAP kinase activation by LPS is inhibited by TcdB [112], but it can also be activated by TcdA or TcdB [93,113,114]. In fact, those conflict results can be attributed to the following differences: cell types, toxin concentrations and sources, assay methods and animal models. So far, all tested cell lines are affected by TcdA and TcdB, but differ in sensitivity [11]. For instance, macrophages and endothelial cells are quite sensitive, while lymphocytes and neutrophils would be much less sensitive. Some early reports showed that TcdA at 10−10 M or TcdB at 10−12 M is able to activate human monocytes as measured by release of interleukin-8 [115,116], while much higher concentration of toxins (10−8~10−9 M) is needed to activate human mast cell line-1 (HMC-1) [117]. Furthermore, TcdB usually has higher cytotoxic potency than TcdA [118]. TcdB is ~2 times more potent than TcdA on HMC-1 cells [119], ~10 times on human colonic epithelial cells [120] and even ~500–1000 times on some other cell lines [49,116]. The source of toxins may also be an important cause of the conflicting results, since C. difficile is a genetically heterogeneous species with substantial chromosomal variation among strains, leading to inherent variability and altered substrate specificity of Rho GTPases, especially in the case of TcdB [71,121,122].
Rho-GTPase activities have been considered to be highly complicated and tightly regulated [123,124,125]. Extensive studies have provided that the activation and signal transduction of the Rho GTPases were regulated by a classic GTPase cycle (Figure 3). This cycle is controlled mainly by three classes of regulatory proteins: (1) guanine nucleotide dissociation inhibitors (GDIs), which extract the inactive Rho GTPase from membranes; (2) guanine nucleotide exchange factors (GEFs), which catalyze nucleotide exchange and mediate activation; and (3) GTPase-activating proteins (GAPs), which stimulate GTP hydrolysis to GDP. Recent investigations have revealed some important regulatory mechanisms of Rho GTPases, including that microRNA (miRNA) regulates post-transcriptional processing of Rho GTPase-encoding mRNAs; palmitoylation and nuclear targeting affect intracellular distribution; post-translational phosphorylation, transglutamination and AMPylation impact Rho-GTPase signaling; ubiquitination controls Rho-GTPase proteins stability and turnover [80]; members of the Rho protein family would regulate each other [85]. These new advances in modes of regulation make the Rho-GTPase signaling network more complicated, but they might also provide new prospect for CDI therapy.
Toxins 07 04874 g003 1024
Figure 3. Schematic diagram of cytopathic effect, cytotoxic effect and immune response induced by C. difficile toxins. After toxins’ endocytosis and delivery, TcdA/B triggers cytopathic and cytotoxic effects in Rho glucosylation-dependent way and -independent way. After infection, the host immune system is activated, leading to activation of the inflammasome and NFκB-mediated pathways. Then, the NFκB-mediated pathways would lead to production of pro-inflammatory cytokines, which contribute to the initiation and propagation of inflammatory response. However, the relationship between Rho glucosylation and the host immune response is unclear.
Figure 3. Schematic diagram of cytopathic effect, cytotoxic effect and immune response induced by C. difficile toxins. After toxins’ endocytosis and delivery, TcdA/B triggers cytopathic and cytotoxic effects in Rho glucosylation-dependent way and -independent way. After infection, the host immune system is activated, leading to activation of the inflammasome and NFκB-mediated pathways. Then, the NFκB-mediated pathways would lead to production of pro-inflammatory cytokines, which contribute to the initiation and propagation of inflammatory response. However, the relationship between Rho glucosylation and the host immune response is unclear.
Toxins 07 04874 g003
In respect of the role of Rho proteins in the toxicity of C. difficile toxins, transfection of Rho GTPases decreases the sensitivity of host cells to TcdB [73,126], while supplementation of glutamine and alanyl-glutamine to TcdA treated cells can increase RhoA expression and then reduce the intestinal epithelial cell damage [127]. Although ADP-ribosylation or phosphorylation of Rho proteins has not been observed in TcdB intoxicated intact oocytes [128], the protective role of phosphorylation of Rho proteins in TcdA and TcdB intoxicated cells has been reported [129,130]. Accumulating evidences have shown that miRNAs are critical in immunity, inflammation and regulation of Rho protein gene expression. Viladomiu and coworkers [131] revealed in 2012 that CDI induces upregulation of miR146b at the gut mucosa, which contributes to pathogenic Th17 responses and impaires immune-regulation. However, the functional roles of miRNAs in colonization, pathogenesis and regulation of the downstream effects of Rho GTPases for C. difficile are almost completely unexplored [132].
In summary, besides the inhibition of effectors coupling and blocking of signal transduction pathways, the glucosylation of Rho GTPases may have influence on other regulation mechanisms of Rho-GTPase activity, which contributes to the final toxicity effects of TcdA and TcdB [53,54]. Further studies are needed to reveal the molecular mechanisms of relationship between CDI and Rho-GTPase regulation, which may be more complicated than what we have known.

Acknowledgments

This work was financially supported by Program for New Century Excellent Talents in University (NCET-10-0399), Innovative Program of Department of Education of Guangdong Province (2013KJCX0013), General Financial Grant from the China Postdoctoral Science Foundation (2012M521593), and Special Financial Grant from the China Postdoctoral Science Foundation (2013T60796).

Author Contributions

Jufang Wang conceived and designed this article. Shuyi Chen and Chunli Sun wrote the manuscript. Shuyi Chen drew all the figures and did all the revise. Haiying Wang reviewed and edited the manuscript. All authors have read and approved the final version of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Bartlett, J.G.; Chang, T.W.; Gurwith, M.; Gorbach, S.L.; Onderdonk, A.B. Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. N. Engl. J. Med. 1978, 298, 531–534. [Google Scholar] [CrossRef] [PubMed]
  2. Planche, T.; Wilcox, M.H. Diagnostic pitfalls in Clostridium difficile infection. Infect. Dis. Clin. N. Am. 2015, 29, 63–82. [Google Scholar] [CrossRef] [PubMed]
  3. Luciano, J.A.; Zuckerbraun, B.S. Clostridium difficile infection: Prevention, treatment, and surgical management. Surg. Clin. N. Am. 2014, 94, 1335–1349. [Google Scholar] [CrossRef] [PubMed]
  4. Bagdasarian, N.; Rao, K.; Malani, P.N. Diagnosis and treatment of Clostridium difficile in adults: A systematic review. JAMA 2015, 313, 398–408. [Google Scholar] [CrossRef] [PubMed]
  5. Pepin, J.; Alary, M.E.; Valiquette, L.; Raiche, E.; Ruel, J.; Fulop, K.; Godin, D.; Bourassa, C. Increasing risk of relapse after treatment of Clostridium difficile colitis in Quebec, Canada. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 2005, 40, 1591–1597. [Google Scholar] [CrossRef] [PubMed]
  6. Vardakas, K.Z.; Polyzos, K.A.; Patouni, K.; Rafailidis, P.I.; Samonis, G.; Falagas, M.E. Treatment failure and recurrence of Clostridium difficile infection following treatment with vancomycin or metronidazole: A systematic review of the evidence. Int. J. Antimicrob. Agents 2012, 40, 1–8. [Google Scholar] [CrossRef] [PubMed]
  7. Shields, K.; Araujo-Castillo, R.V.; Theethira, T.G.; Alonso, C.D.; Kelly, C.P. Recurrent Clostridium difficile infection: From colonization to cure. Anaerobe 2015, 34, 59–73. [Google Scholar] [CrossRef] [PubMed]
  8. Zimlichman, E.; Henderson, D.; Tamir, O.; Franz, C.; Song, P.; Yamin, C.K.; Keohane, C.; Denham, C.R.; Bates, D.W. Health care-associated infections: A meta-analysis of costs and financial impact on the US health care system. JAMA Intern. Med. 2013, 173, 2039–2046. [Google Scholar] [CrossRef] [PubMed]
  9. Kelly, C.P.; LaMont, J.T. Clostridium difficile infection. Ann. Rev. Med. 1998, 49, 375–390. [Google Scholar] [CrossRef] [PubMed]
  10. Voth, D.E.; Ballard, J.D. Clostridium difficile toxins: Mechanism of action and role in disease. Clin. Microbiol. Rev. 2005, 18, 247–263. [Google Scholar] [CrossRef] [PubMed]
  11. Just, I.; Gerhard, R. Large clostridial cytotoxins. Rev. Physiol. Biochem. Pharmacol. 2004, 152, 23–47. [Google Scholar] [PubMed]
  12. Von Eichel-Streiber, C.; Boquet, P.; Sauerborn, M.; Thelestam, M. Large clostridial cytotoxins—A family of glycosyltransferases modifying small GTP-binding proteins. Trends Microbiol. 1996, 4, 375–382. [Google Scholar] [CrossRef]
  13. Schirmer, J.; Aktories, K. Large clostridial cytotoxins: Cellular biology of Rho/Ras-glucosylating toxins. Biochim. Biophys. Acta 2004, 1673, 66–74. [Google Scholar] [CrossRef] [PubMed]
  14. Just, I.; Selzer, J.; Wilm, M.; von Eichel-Streiber, C.; Mann, M.; Aktories, K. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 1995, 375, 500–503. [Google Scholar] [CrossRef] [PubMed]
  15. Just, I.; Wilm, M.; Selzer, J.; Rex, G.; von Eichel-Streiber, C.; Mann, M.; Aktories, K. The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins. J. Biol. Chem. 1995, 270, 13932–13936. [Google Scholar] [CrossRef]
  16. Just, I.; Selzer, J.; Hofmann, F.; Green, G.A.; Aktories, K. Inactivation of Ras by Clostridium sordellii lethal toxin-catalyzed glucosylation. J. Biol. Chem. 1996, 271, 10149–10153. [Google Scholar]
  17. Popoff, M.R.; Chaves-Olarte, E.; Lemichez, E.; von Eichel-Streiber, C.; Thelestam, M.; Chardin, P.; Cussac, D.; Antonny, B.; Chavrier, P.; Flatau, G.; et al. Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation. J. Biol. Chem. 1996, 271, 10217–10224. [Google Scholar]
  18. Selzer, J.; Hofmann, F.; Rex, G.; Wilm, M.; Mann, M.; Just, I.; Aktories, K. Clostridium novyi alpha-toxin-catalyzed incorporation of GlcNAc into Rho subfamily proteins. J. Biol. Chem. 1996, 271, 25173–25177. [Google Scholar] [CrossRef]
  19. Ottlinger, M.E.; Lin, S. Clostridium difficile toxin B induces reorganization of actin, vinculin, and talin in cultured cells. Exp. Cell Res. 1988, 174, 215–229. [Google Scholar] [CrossRef]
  20. Prepens, U.; Just, I.; von Eichel-Streiber, C.; Aktories, K. Inhibition of Fc epsilon-RI-mediated activation of rat basophilic leukemia cells by Clostridium difficile toxin B (monoglucosyltransferase). J. Biol. Chem. 1996, 271, 7324–7329. [Google Scholar]
  21. Schmidt, M.; Rumenapp, U.; Bienek, C.; Keller, J.; von Eichel-Streiber, C.; Jakobs, K.H. Inhibition of receptor signaling to phospholipase D by Clostridium difficile toxin B. Role of Rho proteins. J. Biol. Chem. 1996, 271, 2422–2426. [Google Scholar] [CrossRef]
  22. Subauste, M.C.; von Herrath, M.; Benard, V.; Chamberlain, C.E.; Chuang, T.H.; Chu, K.; Bokoch, G.M.; Hahn, K.M. Rho family proteins modulate rapid apoptosis induced by cytotoxic T lymphocytes and Fas. J. Biol. Chem. 2000, 275, 9725–9733. [Google Scholar] [CrossRef]
  23. Caron, E.; Hall, A. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 1998, 282, 1717–1721. [Google Scholar] [CrossRef] [PubMed]
  24. Jank, T.; Aktories, K. Structure and mode of action of clostridial glucosylating toxins: The ABCD model. Trends Microbiol. 2008, 16, 222–229. [Google Scholar] [CrossRef] [PubMed]
  25. Von Eichel-Streiber, C.; Sauerborn, M.; Kuramitsu, H.K. Evidence for a modular structure of the homologous repetitive C-terminal carbohydrate-binding sites of Clostridium difficile toxins and Streptococcus mutans glucosyltransferases. J. Bacteriol. 1992, 174, 6707–6710. [Google Scholar]
  26. Barroso, L.A.; Moncrief, J.S.; Lyerly, D.M.; Wilkins, T.D. Mutagenesis of the Clostridium difficile toxin B gene and effect on cytotoxic activity. Microb. Pathog. 1994, 16, 297–303. [Google Scholar] [CrossRef] [PubMed]
  27. Busch, C.; Hofmann, F.; Selzer, J.; Munro, S.; Jeckel, D.; Aktories, K. A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins. J. Biol. Chem. 1998, 273, 19566–19572. [Google Scholar] [CrossRef]
  28. Jank, T.; Giesemann, T.; Aktories, K. Clostridium difficile glucosyltransferase toxin B-essential amino acids for substrate binding. J. Biol. Chem. 2007, 282, 35222–35231. [Google Scholar] [CrossRef] [PubMed]
  29. Reineke, J.; Tenzer, S.; Rupnik, M.; Koschinski, A.; Hasselmayer, O.; Schrattenholz, A.; Schild, H.; von Eichel-Streiber, C. Autocatalytic cleavage of Clostridium difficile toxin B. Nature 2007, 446, 415–419. [Google Scholar] [CrossRef] [PubMed]
  30. Egerer, M.; Giesemann, T.; Jank, T.; Satchell, K.J.; Aktories, K. Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity. J. Biol. Chem. 2007, 282, 25314–25321. [Google Scholar] [CrossRef] [PubMed]
  31. Pruitt, R.N.; Chagot, B.; Cover, M.; Chazin, W.J.; Spiller, B.; Lacy, D.B. Structure-function analysis of inositol hexakisphosphate-induced autoprocessing in Clostridium difficile toxin A. J. Biol. Chem. 2009, 284, 21934–21940. [Google Scholar] [CrossRef] [PubMed]
  32. Kreimeyer, I.; Euler, F.; Marckscheffel, A.; Tatge, H.; Pich, A.; Olling, A.; Schwarz, J.; Just, I.; Gerhard, R. Autoproteolytic cleavage mediates cytotoxicity of Clostridium difficile toxin A. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2011, 383, 253–262. [Google Scholar] [CrossRef] [PubMed]
  33. Von Eichel-Streiber, C.; Sauerborn, M. Clostridium difficile toxin A carries a C-terminal repetitive structure homologous to the carbohydrate binding region of streptococcal glycosyltransferases. Gene 1990, 96, 107–113. [Google Scholar] [CrossRef]
  34. Dove, C.H.; Wang, S.Z.; Price, S.B.; Phelps, C.J.; Lyerly, D.M.; Wilkins, T.D.; Johnson, J.L. Molecular characterization of the Clostridium difficile toxin A gene. Infect. Immun. 1990, 58, 480–488. [Google Scholar] [PubMed]
  35. Moncrief, J.S.; Wilkins, T.D. Genetics of Clostridium difficile toxins. Curr. Top. Microbiol. Immunol. 2000, 250, 35–54. [Google Scholar] [PubMed]
  36. Ho, J.G.; Greco, A.; Rupnik, M.; Ng, K.K. Crystal structure of receptor-binding C-terminal repeats from Clostridium difficile toxin A. Proc. Natl. Acad. Sci. USA 2005, 102, 18373–18378. [Google Scholar] [CrossRef] [PubMed]
  37. Greco, A.; Ho, J.G.; Lin, S.J.; Palcic, M.M.; Ng, K.K. Carbohydrate recognition by Clostridium difficile toxin A. Nat. struct. Mol. Biol. 2006, 13, 460–461. [Google Scholar] [CrossRef] [PubMed]
  38. Qa’Dan, M.; Spyres, L.M.; Ballard, J.D. pH-induced conformational changes in Clostridium difficile toxin B. Infect. Immun. 2000, 68, 2470–2474. [Google Scholar] [CrossRef]
  39. Barth, H.; Pfeifer, G.; Hofmann, F.; Maier, E.; Benz, R.; Aktories, K. Low pH-induced formation of ion channels by Clostridium difficile toxin B in target cells. J. Biol. Chem. 2001, 276, 10670–10676. [Google Scholar] [CrossRef] [PubMed]
  40. Pfeifer, G.; Schirmer, J.; Leemhuis, J.; Busch, C.; Meyer, D.K.; Aktories, K.; Barth, H. Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells. J. Biol. Chem. 2003, 278, 44535–44541. [Google Scholar] [CrossRef] [PubMed]
  41. Ziegler, M.O.; Jank, T.; Aktories, K.; Schulz, G.E. Conformational changes and reaction of clostridial glycosylating toxins. J. Mol. Biol. 2008, 377, 1346–1356. [Google Scholar] [CrossRef] [PubMed]
  42. Genisyuerek, S.; Papatheodorou, P.; Guttenberg, G.; Schubert, R.; Benz, R.; Aktories, K. Structural determinants for membrane insertion, pore formation and translocation of Clostridium difficile toxin B. Mol. Microbiol. 2011, 79, 1643–1654. [Google Scholar] [CrossRef] [PubMed]
  43. Rupnik, M.; Pabst, S.; Rupnik, M.; von Eichel-Streiber, C.; Urlaub, H.; Soling, H.D. Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difficile toxin B (TcdB) by host cells. Microbiology 2005, 151, 199–208. [Google Scholar] [CrossRef] [PubMed]
  44. Florin, I.; Thelestam, M. Internalization of Clostridium difficile cytotoxin into cultured human lung fibroblasts. Biochim. Biophys. Acta 1983, 763, 383–392. [Google Scholar] [CrossRef]
  45. Henrigues, B.; Florin, I.; Thelestam, M. Cellular internalisation of Clostridium difficile toxin A. Microb. Pathog. 1987, 2, 455–463. [Google Scholar] [CrossRef]
  46. Etienne-Manneville, S.; Hall, A. Rho GTPases in cell biology. Nature 2002, 420, 629–635. [Google Scholar] [CrossRef] [PubMed]
  47. Burridge, K.; Wennerberg, K. Rho and Rac take center stage. Cell 2004, 116, 167–179. [Google Scholar] [CrossRef]
  48. Jank, T.; Giesemann, T.; Aktories, K. Rho-glucosylating Clostridium difficile toxins A and B: New insights into structure and function. Glycobiology 2007, 17, 15R–22R. [Google Scholar] [CrossRef] [PubMed]
  49. Chaves-Olarte, E.; Weidmann, M.; Eichel-Streiber, C.; Thelestam, M. Toxins A and B from Clostridium difficile differ with respect to enzymatic potencies, cellular substrate specificities, and surface binding to cultured cells. J. Clin. Investig. 1997, 100, 1734–1741. [Google Scholar] [CrossRef] [PubMed]
  50. Mehlig, M.; Moos, M.; Braun, V.; Kalt, B.; Mahony, D.E.; von Eichel-Streiber, C. Variant toxin B and a functional toxin A produced by Clostridium difficile C34. FEMS Microbiol. Lett. 2001, 198, 171–176. [Google Scholar] [CrossRef] [PubMed]
  51. Genth, H.; Pauillac, S.; Schelle, I.; Bouvet, P.; Bouchier, C.; Varela-Chavez, C.; Just, I.; Popoff, M.R. Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: Molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins. Cell Microbiol. 2014, 16, 1706–1721. [Google Scholar] [CrossRef] [PubMed]
  52. Vetter, I.R.; Wittinghofer, A. The guanine nucleotide-binding switch in three dimensions. Science 2001, 294, 1299–1304. [Google Scholar] [CrossRef] [PubMed]
  53. Herrmann, C.; Ahmadian, M.R.; Hofmann, F.; Just, I. Functional consequences of monoglucosylation of Ha-Ras at effector domain amino acid threonine 35. J. Biol. Chem. 1998, 273, 16134–16139. [Google Scholar] [CrossRef] [PubMed]
  54. Sehr, P.; Joseph, G.; Genth, H.; Just, I.; Pick, E.; Aktories, K. Glucosylation and ADP ribosylation of rho proteins: Effects on nucleotide binding, GTPase activity, and effector coupling. Biochemistry 1998, 37, 5296–5304. [Google Scholar] [CrossRef] [PubMed]
  55. Genth, H.; Dreger, S.C.; Huelsenbeck, J.; Just, I. Clostridium difficile toxins: More than mere inhibitors of Rho proteins. Int. J. Biochem. Cell Biol. 2008, 40, 592–597. [Google Scholar] [CrossRef] [PubMed]
  56. Reinert, D.J.; Jank, T.; Aktories, K.; Schulz, G.E. Structural basis for the function of Clostridium difficile toxin B. J. Mol. Biol. 2005, 351, 973–981. [Google Scholar] [CrossRef] [PubMed]
  57. Unligil, U.M.; Rini, J.M. Glycosyltransferase structure and mechanism. Curr. Opin. Struct. Biol. 2000, 10, 510–517. [Google Scholar] [CrossRef]
  58. Qasba, P.K.; Ramakrishnan, B.; Boeggeman, E. Substrate-induced conformational changes in glycosyltransferases. Trends Biochem. Sci. 2005, 30, 53–62. [Google Scholar] [CrossRef] [PubMed]
  59. Jank, T.; Pack, U.; Giesemann, T.; Schmidt, G.; Aktories, K. Exchange of a single amino acid switches the substrate properties of RhoA and RhoD toward glucosylating and transglutaminating toxins. J. Biol. Chem. 2006, 281, 19527–19535. [Google Scholar] [CrossRef] [PubMed]
  60. Müller, S.; von Eichel-Streiber, C.; Moos, M. Impact of amino acids 22–27 of Rho-subfamily GTPases on glucosylation by the large clostridial cytotoxins TcsL-1522, TcdB-1470 and TcdB-8864. Eur. J. Biochem. 1999, 266, 1073–1080. [Google Scholar] [CrossRef] [PubMed]
  61. Genth, H.; Aktories, K.; Just, I. Monoglucosylation of RhoA at threonine 37 blocks cytosol-membrane cycling. J. Biol. Chem. 1999, 274, 29050–29056. [Google Scholar] [CrossRef] [PubMed]
  62. Kozma, R.; Ahmed, S.; Best, A.; Lim, L. The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol. Cell. Biol. 1995, 15, 1942–1952. [Google Scholar] [CrossRef] [PubMed]
  63. Nobes, C.D.; Hall, A. Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995, 81, 53–62. [Google Scholar] [CrossRef]
  64. Gerhard, R.; Schmidt, G.; Hofmann, F.; Aktories, K. Activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor 1 increases intestinal permeability in Caco-2 cells. Infect. Immun. 1998, 66, 5125–5131. [Google Scholar] [PubMed]
  65. Hecht, G.; Pothoulakis, C.; LaMont, J.T.; Madara, J.L. Clostridium difficile toxin A perturbs cytoskeletal structure and tight juction permeability of cultured human intestinal epithelial monolayers. J. Clin. Investig. 1988, 82, 1516–1524. [Google Scholar] [CrossRef] [PubMed]
  66. Hecht, G.; Koutsouris, A.; Pothoulakis, C.; LaMont, J.T.; Madara, J.L. Clostridium difficile toxin B disrupts the barrier function of T84 monolayers. Gastroenterology 1992, 102, 416–423. [Google Scholar] [PubMed]
  67. Johal, S.S.; Solomon, K.; Dodson, S.; Borriello, P.; Mahida, Y.R. Differential effects of varying concentrations of Clostridium difficile toxin A on epithelial barrier function and expression of cytokines. J. Infect. Dis. 2004, 189, 2110–2119. [Google Scholar] [CrossRef] [PubMed]
  68. Moore, R.; Pothoulakis, C.; LaMont, J.T.; Carlson, S.; Madara, J.L. C. difficile toxin A increases intestinal permeability and induces Cl-secretion. Am. J. Physiol. 1990, 259, G165–G172. [Google Scholar] [PubMed]
  69. Triadafilopoulos, G.; Pothoulakis, C.; O’Brien, M.J.; LaMont, J.T. Differential effects of Clostridium difficile toxins A and B on rabbit ileum. Gastroenterology 1987, 93, 273–279. [Google Scholar] [PubMed]
  70. Triadafilopoulos, G.; Pothoulakis, C.; Weiss, R.; Tiampaolo, C.; LaMont, J.T. Comparative study of Clostridium difficile toxin A and cholera toxin in rabbit ileum. Gastroenterology 1989, 97, 1186–1192. [Google Scholar] [PubMed]
  71. Chaves-Olarte, E.; Freer, E.; Parra, A.; Guzman-Verri, C.; Moreno, E.; Thelestam, M. R-Ras glucosylation and transient RhoA activation determine the cytopathic effect produced by toxin B variants from toxin A-negative strains of Clostridium difficile. J. Biol. Chem. 2003, 278, 7956–7963. [Google Scholar] [CrossRef] [PubMed]
  72. Chen, M.L.; Pothoulakis, C.; LaMont, J.T. Protein kinase C signaling regulates ZO-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile toxin A. J. Biol. Chem. 2002, 277, 4247–4254. [Google Scholar] [CrossRef] [PubMed]
  73. Kim, H.; Rhee, S.H.; Pothoulakis, C.; LaMont, J.T. Clostridium difficile toxin A binds colonocyte Src causing dephosphorylation of focal adhesion kinase and paxillin. Exp. Cell Res. 2009, 315, 3336–3344. [Google Scholar] [CrossRef] [PubMed]
  74. Halabi-Cabezon, I.; Huelsenbeck, J.; May, M.; Ladwein, M.; Rottner, K.; Just, I.; Genth, H. Prevention of the cytopathic effect induced by Clostridium difficile Toxin B by active Rac1. FEBS Lett. 2008, 582, 3751–3756. [Google Scholar] [CrossRef] [PubMed]
  75. Huelsenbeck, J.; Dreger, S.C.; Gerhard, R.; Fritz, G.; Just, I.; Genth, H. Upregulation of the immediate early gene product RhoB by exoenzyme C3 from Clostridium limosum and toxin B from Clostridium difficile. Biochemistry 2007, 46, 4923–4931. [Google Scholar] [CrossRef] [PubMed]
  76. Gerhard, R.; Tatge, H.; Genth, H.; Thum, T.; Borlak, J.; Fritz, G.; Just, I. Clostridium difficile toxin A induces expression of the stress-induced early gene product RhoB. J. Biol. Chem. 2005, 280, 1499–1505. [Google Scholar] [CrossRef] [PubMed]
  77. Genth, H.; Huelsenbeck, J.; Hartmann, B.; Hofmann, F.; Just, I.; Gerhard, R. Cellular stability of Rho-GTPases glucosylated by Clostridium difficile toxin B. FEBS Lett. 2006, 580, 3565–3569. [Google Scholar] [CrossRef] [PubMed]
  78. Mahida, Y.R.; Galvin, A.; Makh, S.; Hyde, S.; Sanfilippo, L.; Borriello, S.P.; Sewell, H.F. Effect of Clostridium difficile Toxin A on Human Colonic Lamina Propria Cells: Early Loss of Macrophages Followed by T-Cell Apoptosis. Infect. Immun. 1998, 66, 5462–5469. [Google Scholar] [PubMed]
  79. Gerhard, R.; Nottrott, S.; Schoentaube, J.; Tatge, H.; Olling, A.; Just, I. Glucosylation of Rho GTPases by Clostridium difficile toxin A triggers apoptosis in intestinal epithelial cells. J. Med. Microbiol. 2008, 57, 765–770. [Google Scholar] [CrossRef] [PubMed]
  80. Brito, G.A.; Fujji, J.; Carneiro-Filho, B.A.; Lima, A.A.; Obrig, T.; Guerrant, R.L. Mechanism of Clostridium difficile toxin A-induced apoptosis in T84 cells. J. Infect. Dis. 2002, 186, 1438–1447. [Google Scholar] [CrossRef] [PubMed]
  81. Hippenstiel, S.; Schmeck, B.; N’Guessan, P.D.; Seybold, J.; Krull, M.; Preissner, K.; Eichel-Streiber, C.V.; Suttorp, N. Rho protein inactivation induced apoptosis of cultured human endothelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 2002, 283, L830–L838. [Google Scholar] [CrossRef] [PubMed]
  82. Teichert, M.; Tatge, H.; Schoentaube, J.; Just, I.; Gerhard, R. Application of mutated Clostridium difficile toxin A for determination of glucosyltransferase-dependent effects. Infect. Immun. 2006, 74, 6006–6010. [Google Scholar] [CrossRef] [PubMed]
  83. Nottrott, S.; Schoentaube, J.; Genth, H.; Just, I.; Gerhard, R. Clostridium difficile toxin A-induced apoptosis is p53-independent but depends on glucosylation of Rho GTPases. Apoptosis Int. J. Program. Cell Death 2007, 12, 1443–1453. [Google Scholar] [CrossRef] [PubMed]
  84. Huelsenbeck, J.; Dreger, S.; Gerhard, R.; Barth, H.; Just, I.; Genth, H. Difference in the cytotoxic effects of toxin B from Clostridium difficile strain VPI 10463 and toxin B from variant Clostridium difficile strain 1470. Infect. Immun. 2007, 75, 801–809. [Google Scholar] [CrossRef] [PubMed]
  85. Wang, H.; Sun, X.; Zhang, Y.; Li, S.; Chen, K.; Shi, L.; Nie, W.; Kumar, R.; Tzipori, S.; Wang, J.; et al. A chimeric toxin vaccine protects against primary and recurrent Clostridium difficile infection. Infect. Immun. 2012, 80, 2678–2688. [Google Scholar] [CrossRef] [PubMed]
  86. Sun, C.; Wang, H.; Chen, S.; Li, Z.; Li, S.; Wang, J. Recombinant Clostridium difficile toxin B induces endoplasmic reticulum stress in mouse colonal carcinoma cells. Acta Biochim. Biophys. Sin. 2014, 46, 973–981. [Google Scholar] [CrossRef] [PubMed]
  87. Chumbler, N.M.; Farrow, M.A.; Lapierre, L.A.; Franklin, J.L.; Haslam, D.B.; Goldenring, J.R.; Lacy, D.B. Clostridium difficile Toxin B causes epithelial cell necrosis through an autoprocessing-independent mechanism. PLoS Pathog. 2012. [Google Scholar] [CrossRef]
  88. Jafari, N.V.; Kuehne, S.A.; Bryant, C.E.; Elawad, M.; Wren, B.W.; Minton, N.P.; Allan, E.; Bajaj-Elliott, M. Clostridium difficile modulates host innate immunity via toxin-independent and dependent mechanism(s). PLoS ONE 2013. [Google Scholar] [CrossRef] [PubMed]
  89. Solomon, K. The host immune response to Clostridium difficile infection. Ther. Adv. Infect. Dis. 2013, 1, 19–35. [Google Scholar] [CrossRef] [PubMed]
  90. Kelly, C.P.; Pothoulakis, C.; LaMont, J.T. Clostridium difficile colitis. N. Engl. J. Med. 1994, 330, 257–262. [Google Scholar] [CrossRef] [PubMed]
  91. Xu, H.; Yang, J.; Gao, W.; Li, L.; Li, P.; Zhang, L.; Gong, Y.N.; Peng, X.; Xi, J.J.; Chen, S.; et al. Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome. Nature 2014, 513, 237–241. [Google Scholar] [CrossRef] [PubMed]
  92. Ng, J.; Hirota, S.A.; Gross, O.; Li, Y.; Ulke-Lemee, A.; Potentier, M.S.; Schenck, L.P.; Vilaysane, A.; Seamone, M.E.; Feng, H.; et al. Clostridium difficile toxin-induced inflammation and intestinal injury are mediated by the inflammasome. Gastroenterology 2010, 139, 542–552. [Google Scholar] [CrossRef] [PubMed]
  93. Warny, M.; Keates, A.C.; Keates, S.; Castagliuolo, I.; Zacks, J.K.; Aboudola, S.; Qamar, A.; Pothoulakis, C.; LaMont, J.T.; Kelly, C.P. p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis. J. Clin. Investig. 2000, 105, 1147–1156. [Google Scholar] [CrossRef] [PubMed]
  94. Yeh, C.Y.; Lin, C.N.; Chang, C.F.; Lin, C.H.; Lien, H.T.; Chen, J.Y.; Chia, J.S. C-terminal repeats of Clostridium difficile toxin A induce production of chemokine and adhesion molecules in endothelial cells and promote migration of leukocytes. Infect. Immun. 2008, 76, 1170–1178. [Google Scholar] [CrossRef] [PubMed]
  95. Sun, X.; He, X.; Tzipori, S.; Gerhard, R.; Feng, H. Essential role of the glucosyltransferase activity in Clostridium difficile toxin-induced secretion of TNF-alpha by macrophages. Microb. Pathog. 2009, 46, 298–305. [Google Scholar] [CrossRef] [PubMed]
  96. Castagliuolo, I.; Keates, A.C.; Wang, C.C.; Pasha, A.; Valenick, L.; Kelly, C.P.; Nikulasson, S.T.; LaMont, J.T.; Pothoulakis, C. Clostridium difficile toxin A stimulates macrophage-inflammatory protein-2 production in rat intestinal epithelial cells. J. Immunol. 1998, 160, 6039–6045. [Google Scholar] [PubMed]
  97. Kim, J.M.; Lee, J.Y.; Yoon, Y.M.; Oh, Y.K.; Youn, J.; Kim, Y.J. NF-kappa B activation pathway is essential for the chemokine expression in intestinal epithelial cells stimulated with Clostridium difficile toxin A. Scand. J. Immunol. 2006, 63, 453–460. [Google Scholar] [CrossRef] [PubMed]
  98. Jefferson, K.K.; Smith, M.F., Jr.; Bobak, D.A. Roles of intracellular calcium and NF-kappa B in the Clostridium difficile toxin A-induced up-regulation and secretion of IL-8 from human monocytes. J. Immunol. 1999, 163, 5183–5191. [Google Scholar] [PubMed]
  99. Yoshino, Y.; Kitazawa, T.; Ikeda, M.; Tatsuno, K.; Yanagimoto, S.; Okugawa, S.; Yotsuyanagi, H.; Ota, Y. Clostridium difficile flagellin stimulates toll-like receptor 5, and toxin B promotes flagellin-induced chemokine production via TLR5. Life Sci. 2013, 92, 211–217. [Google Scholar] [CrossRef] [PubMed]
  100. Huang, T.; Li, S.; Li, G.; Tian, Y.; Wang, H.; Shi, L.; Perez-Cordon, G.; Mao, L.; Wang, X.; Wang, J.; et al. Utility of Clostridium difficile toxin B for inducing anti-tumor immunity. PLoS ONE 2014, 9, e110826. [Google Scholar] [CrossRef] [PubMed]
  101. Mizuno, T.; Kaibuchi, K.; Ando, S.; Musha, T.; Hiraoka, K.; Takaishi, K.; Asada, M.; Nunoi, H.; Matsuda, I.; Takai, Y. Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins. J. Biol. Chem. 1992, 267, 10215–10218. [Google Scholar] [PubMed]
  102. Heyworth, P.G.; Knaus, U.G.; Xu, X.; Uhlinger, D.J.; Conroy, L.; Bokoch, G.M.; Curnutte, J.T. Requirement for posttranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase. Mol. Biol. Cell 1993, 4, 261–269. [Google Scholar] [CrossRef]
  103. Sundaresan, M.; Yu, Z.X.; Ferrans, V.J.; Sulciner, D.J.; Gutkind, J.S.; Irani, K.; Goldschmidt-Clermont, P.J.; Finkel, T. Regulation of reactive-oxygen-species generation in fibroblasts by Rac1. Biochem. J. 1996, 318, 379–382. [Google Scholar] [CrossRef] [PubMed]
  104. Herkert, O.; Diebold, I.; Brandes, R.P.; Hess, J.; Busse, R.; Gorlach, A. NADPH oxidase mediates tissue factor-dependent surface procoagulant activity by thrombin in human vascular smooth muscle cells. Circulation 2002, 105, 2030–2036. [Google Scholar] [CrossRef] [PubMed]
  105. Qiu, B.; Pothoulakis, C.; Castagliuolo, I.; Nikulasson, S.; LaMont, J.T. Participation of reactive oxygen metabolites in Clostridium difficile toxin A-induced enteritis in rats. Am. J. Physiol. 1999, 276, G485–G490. [Google Scholar] [PubMed]
  106. Farrow, M.A.; Chumbler, N.M.; Lapierre, L.A.; Franklin, J.L.; Rutherford, S.A.; Goldenring, J.R.; Lacy, D.B. Clostridium difficile toxin B-induced necrosis is mediated by the host epithelial cell NADPH oxidase complex. Proc. Natl. Acad. Sci. USA 2013, 110, 18674–18679. [Google Scholar] [CrossRef] [PubMed]
  107. He, D.; Sougioultzis, S.; Hagen, S.; Liu, J.; Keates, S.; Keates, A.C.; Pothoulakis, C.; Lamont, J.T. Clostridium difficile toxin A triggers human colonocyte IL-8 release via mitochondrial oxygen radical generation. Gastroenterology 2002, 122, 1048–1057. [Google Scholar] [CrossRef] [PubMed]
  108. Arbibe, L.; Mira, J.P.; Teusch, N.; Kline, L.; Guha, M.; Mackman, N.; Godowski, P.J.; Ulevitch, R.J.; Knaus, U.G. Toll-like receptor 2-mediated NF-kappa B activation requires a Rac1-dependent pathway. Nat. Immunol. 2000, 1, 533–540. [Google Scholar] [CrossRef] [PubMed]
  109. Teusch, N.; Lombardo, E.; Eddleston, J.; Knaus, U.G. The low molecular weight GTPase RhoA and atypical protein kinase Czeta are required for TLR2-mediated gene transcription. J. Immunol. 2004, 173, 507–514. [Google Scholar] [CrossRef] [PubMed]
  110. Huang, S.; Chen, L.Y.; Zuraw, B.L.; Ye, R.D.; Pan, Z.K. Chemoattractant-stimulated NF-kappaB activation is dependent on the low molecular weight GTPase RhoA. J. Biol. Chem. 2001, 276, 40977–40981. [Google Scholar] [CrossRef] [PubMed]
  111. Shen, A. Clostridium difficile toxins: Mediators of inflammation. J. Innate Immun. 2012, 4, 149–158. [Google Scholar] [CrossRef] [PubMed]
  112. Hippenstiel, S.; Soeth, S.; Kellas, B.; Fuhrmann, O.; Seybold, J.; Krull, M.; Eichel-Streiber, C.; Goebeler, M.; Ludwig, S.; Suttorp, N. Rho proteins and the p38-MAPK pathway are important mediators for LPS-induced interleukin-8 expression in human endothelial cells. Blood 2000, 95, 3044–3051. [Google Scholar] [PubMed]
  113. Kim, H.; Kokkotou, E.; Na, X.; Rhee, S.H.; Moyer, M.P.; Pothoulakis, C.; Lamont, J.T. Clostridium difficile toxin A-induced colonocyte apoptosis involves p53-dependent p21(WAF1/CIP1) induction via p38 mitogen-activated protein kinase. Gastroenterology 2005, 129, 1875–1888. [Google Scholar] [CrossRef] [PubMed]
  114. Kim, H.; Rhee, S.H.; Kokkotou, E.; Na, X.; Savidge, T.; Moyer, M.P.; Pothoulakis, C.; LaMont, J.T. Clostridium difficile toxin A regulates inducible cyclooxygenase-2 and prostaglandin E2 synthesis in colonocytes via reactive oxygen species and activation of p38 MAPK. J. Biol. Chem. 2005, 280, 21237–21245. [Google Scholar] [CrossRef] [PubMed]
  115. Linevsky, J.K.; Pothoulakis, C.; Keates, S.; Warny, M.; Keates, A.C.; Lamont, J.T.; Kelly, C.P. IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes. Am. Physiol. Soc. 1997, 273, G1333–G1340. [Google Scholar]
  116. Flegel, W.A.; Müller, F.; Däubener, W.; Fischer, H.G.; Hadding, U.; Northoff, H. Cytokine response by human monocytes to Clostridium difficile toxin A and toxin B. Infect. Immun. 1991, 59, 3659–3666. [Google Scholar] [PubMed]
  117. Balletta, A.; Lorenz, D.; Rummel, A.; Gerhard, R.; Bigalke, H.; Wegner, F. Clostridium difficile toxin B inhibits the secretory response of human mast cell ling-1 (HMC-1) cells stimulated with high free-Ca2+ and GTPγs. Toxicology 2014, 328, 48–56. [Google Scholar] [CrossRef] [PubMed]
  118. Lyerly, D.M.; Lockwood, D.E.; Richardson, S.H.; Wilkins, T.D. Biological activities of toxins A and B of Clostridium difficile. Infect. Immun. 1982, 35, 1147–1150. [Google Scholar] [PubMed]
  119. Meyer, G.K.; Neetz, A.; Brandes, G.; Tsikas, D.; Butterfield, J.H.; Just, I.; Gerhard, R. Clostridium difficile toxins A and B directly stimulate human mast cells. Infect. Immun. 2007, 75, 3868–3876. [Google Scholar] [CrossRef] [PubMed]
  120. Riegler, M.; Sedivy, R.; Pothoulakis, C.; Hamilton, G.; Zacherl, J.; Bischof, G.; Cosentini, E.; Feil, W.; Schiessel, R.; LaMont, J.T.; et al. Clostridium difficile toxin B is more potent than toxin A in damaging human colonic epithelium in virto. J. Clin. Investig. 1995, 95, 2004–2011. [Google Scholar] [CrossRef] [PubMed]
  121. Stabler, R.A.; Gerding, D.N.; Songer, J.G.; Drudy, D.; Brazier, J.S.; Trinh, H.T.; Witney, A.A.; Hinds, J.; Wren, B.W. Comparative phylogenomics of Clostridium difficile reveals clade specificity and microevolution of hypervirulent strains. J. Bacteriol. 2006, 188, 7297–7305. [Google Scholar] [CrossRef] [PubMed]
  122. Alfa, M.J.; Kabani, A.; Lyerly, D.; Moncrief, S.; Neville, L.M.; Al-Barrak, A.; Harding, G.K.; Dyck, B.; Olekson, K.; Embil, J.M. Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile responsible for a nosocomial outbreak of Clostridium difficile-associated diarrhea. J. Clin. Microbiol. 2000, 38, 2706–2714. [Google Scholar] [PubMed]
  123. Liu, M.; Bi, F.; Zhou, X.; Zheng, Y. Rho GTPase regulation by miRNAs and covalent modifications. Trends Cell Biol. 2012, 22, 365–373. [Google Scholar] [CrossRef] [PubMed]
  124. Visvikis, O.; Maddugoda, M.P.; Lemichez, E. Direct modifications of Rho proteins: Deconstructing GTPase regulation. Biol. Cell 2010, 102, 377–389. [Google Scholar] [CrossRef] [PubMed]
  125. Guilluy, C.; Garcia-Mata, R.; Burridge, K. Rho protein crosstalk: Another social network? Trends Cell Biol. 2011, 21, 718–726. [Google Scholar] [CrossRef] [PubMed]
  126. Giry, M.; Popoff, M.R.; von Eichel-Streiber, C.; Boquet, P. Transient expression of RhoA, -B, and -C GTPases in HeLa cells potentiates resistance to Clostridium difficile toxins A and B but not to Clostridium sordellii lethal toxin. Infect. Immun. 1995, 63, 4063–4071. [Google Scholar] [PubMed]
  127. Santos, A.A.; Braga-Neto, M.B.; Oliveira, M.R.; Freire, R.S.; Barros, E.B.; Santiago, T.M.; Rebelo, L.M.; Mermelstein, C.; Warren, C.A.; Guerrant, R.L.; et al. Glutamine and alanyl-glutamine increase RhoA expression and reduce Clostridium difficile toxin-a-induced intestinal epithelial cell damage. BioMed Res. Int. 2013. [Google Scholar] [CrossRef] [PubMed]
  128. Just, I.; Richter, H.P.; Prepens, U.; von Eichel-Streiber, C.; Aktories, K. Probing the action of Clostridium difficile toxin B in Xenopus laevis oocytes. J. Cell Sci. 1994, 107, 1653–1659. [Google Scholar] [PubMed]
  129. Brandes, V.; Schelle, I.; Brinkmann, S.; Schulz, F.; Schwarz, J.; Gerhard, R.; Genth, H. Protection from Clostridium difficile toxin B-catalysed Rac1/Cdc42 glucosylation by tauroursodeoxycholic acid-induced Rac1/Cdc42 phosphorylation. Biol. Chem. 2012, 393, 77–84. [Google Scholar] [CrossRef] [PubMed]
  130. Schoentaube, J.; Olling, A.; Tatge, H.; Just, I.; Gerhard, R. Serine-71 phosphorylation of Rac1/Cdc42 diminishes the pathogenic effect of Clostridium difficile toxin A. Cell. Microbiol. 2009, 11, 1816–1826. [Google Scholar] [CrossRef] [PubMed]
  131. Viladomiu, M.; Hontecillas, R.; Pedragosa, M.; Carbo, A.; Hoops, S.; Michalak, P.; Michalak, K.; Guerrant, R.L.; Roche, J.K.; Warren, C.A.; et al. Modeling the role of peroxisome proliferator-activated receptor gamma and microRNA-146 in mucosal immune responses to Clostridium difficile. PLoS ONE 2012. [Google Scholar] [CrossRef] [PubMed]
  132. Mraheil, M.A.; Billion, A.; Kuenne, C.; Pischimarov, J.; Kreikemeyer, B.; Engelmann, S.; Hartke, A.; Giard, J.C.; Rupnik, M.; Vorwerk, S.; et al. Comparative genome-wide analysis of small RNAs of major Gram-positive pathogens: From identification to application. Microb. Biotechnol. 2010, 3, 658–676. [Google Scholar] [CrossRef] [PubMed]

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MDPI and ACS Style

Chen, S.; Sun, C.; Wang, H.; Wang, J. The Role of Rho GTPases in Toxicity of Clostridium difficile Toxins. Toxins 2015, 7, 5254-5267. https://doi.org/10.3390/toxins7124874

AMA Style

Chen S, Sun C, Wang H, Wang J. The Role of Rho GTPases in Toxicity of Clostridium difficile Toxins. Toxins. 2015; 7(12):5254-5267. https://doi.org/10.3390/toxins7124874

Chicago/Turabian Style

Chen, Shuyi, Chunli Sun, Haiying Wang, and Jufang Wang. 2015. "The Role of Rho GTPases in Toxicity of Clostridium difficile Toxins" Toxins 7, no. 12: 5254-5267. https://doi.org/10.3390/toxins7124874

APA Style

Chen, S., Sun, C., Wang, H., & Wang, J. (2015). The Role of Rho GTPases in Toxicity of Clostridium difficile Toxins. Toxins, 7(12), 5254-5267. https://doi.org/10.3390/toxins7124874

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