Exploiting cell death pathways by an E. coli cytotoxin: autophagy as a double-edged sword for the host

Cytotoxic necrotizing factor 1 is a bacterial protein toxin from Escherichia coli that is able to activate the Rho GTPases and to hinder apoptosis and mitotic catastrophe. Upon exposure to toxin, cells undergo a complex framework of changes, including cytoskeleton remodeling and multinucleation. These cells also show a high survival rate for long periods of time and improve both their macropinocytotic scavenging activities and microautophagy. Only at the very end, probably when "feeding" materials are exhausted, do these cells die by autophagy. Taking into account the complex role of bacterial protein toxins in the infectious processes, we indicate the CNF1 activity as a Janus-faced paradigm of those bacteria that hijack cell fate to their own benefit. This could somehow be linked to the hypothesized connection between certain bacterial toxins and cancer onset.     

Insertion-trigger residues differentially modulate endosomal escape by cytotoxic necrotizing factor toxins

The cellular specificity, potency, and modular nature of bacterial protein toxins enable their application for targeted cytosolic delivery of therapeutic cargo. Efficient endosomal escape is a critical step in the design of bacterial toxin-inspired drug delivery (BTIDD) vehicles to avoid lysosomal degradation and promote optimal cargo delivery. The cytotoxic necrotizing factor (CNF) family of modular toxins represents a useful model for investigating cargo-delivery mechanisms due to the availability of many homologs with high sequence identity, their flexibility in swapping domains, and their differential activity profiles. Previously, we found that CNFy is more sensitive to endosomal acidification inhibitors than CNF1 and CNF2. Here, we report that CNF3 is even less sensitive than CNF1/2. We identified two amino acid residues within the putative translocation domain (E374 and E412 in CNFy, Q373 and S411 in CNF3) that differentiate between these two toxins. Swapping these corresponding residues in each toxin changed the sensitivity to endosomal acidification and efficiency of cargo-delivery to be more similar to the other toxin. Results suggested that trafficking to the more acidic late endosome is required for cargo delivery by CNFy but not CNF3. This model was supported by results from toxin treatment of cells in the presence of NH₄Cl, which blocks endosomal acidification, and of small-molecule inhibitors EGA, which blocks trafficking to late endosomes, and ABMA, which blocks endosomal escape and trafficking to the lysosomal degradative pathway. These findings suggest that it is possible to fine-tune endosomal escape and cytosolic cargo delivery efficiency in designing BTIDD platforms.     

CNF and DNT

The actin cytoskeleton of mammalian cells is involved in many processes that affect the growth and colonization of bacteria, such as migration of immune cells, phagocytosis by macrophages, secretion of cytokines, maintenance of epithelial barrier functions and others. With respect to these functions, it is not surprising that many bacterial protein toxins, which are important virulence factors and causative agents of human and/or animal diseases, target the actin cytoskeleton of the host. Some toxins target actin directly, such as the C2 toxin produced by Clostridium botulinum. Moreover, bacterial toxins target the cytoskeleton indirectly by modifying actin regulators such as the low-molecular-mass guanosine triphosphate (GTP)-binding proteins of the Rho family. Remarkably, toxins affect these GTPases in a bidirectional manner. Some toxins inhibit and some activate the GTPases. Here we review the Rho-activating toxins CNF1 and CNF2 (cytotoxic necrotizing factors) from Escherichia coli, the Yersinia CNF_Y and the dermonecrotic toxin (DNT) from Bordetella species. We describe and compare their uptake into mammalian cells, mode of action, structure–function relationship, substrate specificity and role in diseases.     

Modification of plant Rac/Rop GTPase signalling using bacterial toxin transgenes

Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co‐expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive‐like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co‐expressed constitutively active Rop4, blocking the hypersensitive response caused by over‐expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop‐dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop‐dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.     

Structure of the Rho-activating domain of Escherichia coli cytotoxic necrotizing factor 1.

Certain uropathogenic and neonatal meningitis-causing strains of Escherichia coli express a 114 kDa protein toxin called cytotoxic necrotizing factor 1 (CNF1). The toxin causes alteration of the host cell actin cytoskeleton and promotes bacterial invasion of blood-brain barrier endothelial cells. CNF1 belongs to a unique group of large cytotoxins that cause constitutive activation of Rho guanosine triphosphatases (GTPases), which are key regulators of the actin cytoskeleton. This group also includes E. coli cytotoxic necrotizing factor 2 (CNF2, 114 kDa) and dermonecrotic toxins (DNT, 159 kDa) of Bordetella spp. with related sequences occurring in Yersinia spp. Here we show that the catalytic region of CNF1 exhibits a novel protein fold as determined by its 1.83 A resolution crystal structure. The structure reveals that CNF1 has a Cys-His-main chain oxygen catalytic triad reminiscent of enzymes belonging to the catalytic triad superfamily. The position of the catalytic Cys residue at the base of a deep pocket restricts access to potential substrates and helps explain the high specificity of this and related toxins.     

Exchange of a single amino acid switches the substrate properties of RhoA and RhoD toward glucosylating and transglutaminating toxins

Rho GTPases are the preferred targets of various bacterial cytotoxins, including Clostridium difficile toxins A and B, Clostridium sordellii lethal toxin, the cytotoxic necrotizing factors (CNF1) from Escherichia coli, and the dermonecrotizing toxin (DNT) from Bordetella species. The toxins inactivate or activate specific sets of Rho GTPases by mono-O-glucosylation and deamidation/transglutamination, respectively. Here we studied the structural basis of the recognition of RhoA, which is modified by toxin B, CNF1, and DNT, in comparison with RhoD, which is solely a substrate for lethal toxin. We found that a single amino acid residue in RhoA and RhoD defines the substrate specificity for toxin B and lethal toxin. Change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin. Accordingly, change of the equivalently positioned phenylalanine 85 in RhoD with serine allowed glucosylation by toxin B. Comparable results were achieved with the Rho-activating and transglutaminating enzymes CNF1 and DNT. Here, amino acid glutamate 64 of RhoA and the equivalent aspartate 76 of RhoD define substrate specificity for CNF1 and DNT, respectively. These data indicate that single amino acid residues located in the switch II region of Rho proteins determine enzyme specificity for diverse bacterial toxins.     

A single amino acid substitution in the enzymatic domain of cytotoxic necrotizing factor type 1 of Escherichia coli alters the tissue culture phenotype to that of the dermonecrotic toxin of Bordetella spp

Cytotoxic necrotizing factor type 1 (CNF1) and dermonecrotic toxin (DNT) share homology within their catalytic domains and possess deamidase and transglutaminase activities. Although each toxin has a preferred enzymatic activity (i.e. deamidation for CNF1 and transglutamination for DNT) as well as target substrates, both modify a specific glutamine residue in RhoA, Rac1 and Cdc42, which renders these GTPases constitutively active. Here we show that despite their similar mechanisms of action CNF1 and DNT induced unique phenotypes on HEp-2 and Swiss 3T3 cells. CNF1 induced multinucleation of HEp-2 cells and was cytotoxic for Swiss 3T3 cells (with binucleation of the few surviving cells) while DNT showed no morphological effects on HEp-2 cells but did induce binucleation of Swiss 3T3 cells. To determine if the enzymatic domain of each toxin dictated the induced phenotype, we constructed enzymatically active chimeric toxins and mutant toxins that contained single amino acid substitutions within the catalytic site and tested these molecules in tissue culture and enzymatic assays. Moreover, both site-directed mutant toxins showed reduced time to maximum transglutamination of RhoA compared with the parent toxins. Nevertheless, the substitution of threonine for Lys(1310) in the DNT-based mutant, while affecting transglutamination efficiency of the toxin, did not abrogate that enzymatic activity.     

The p21 Rho-activating toxin cytotoxic necrotizing factor 1 is endocytosed by a clathrin-independent mechanism and enters the cytosol by an acidic-dependent membrane translocation step

Cytotoxic necrotizing factor 1 (CNF1), a protein produced by pathogenic strains of Escherichia coli, activates the p21 Rho-GTP-binding protein, inducing a profound reorganization of the actin cytoskeleton. CNF1 binds to its cell surface receptor on HEp-2 cells with high affinity (K(d) = 20 pM). In HEp-2 cells the action of CNF1 is not blocked in the presence of filipin, a drug described to reduce cholera toxin internalization by the caveolae-like mechanism. Moreover, HEp-2 cells, which express a dominant negative form of proteins that impair the formation of clathrin coated-vesicles and internalization of transferrin (Eps15, dynamin or intersectin-Src homology 3), are still sensitive to CNF1. In this respect, the endocytosis of CNF1 is similar to the plant toxin ricin. However, unlike ricin toxin, CNF1 does not cross the Golgi apparatus and requires an acidic cell compartment to transfer its enzymatic activity into the cytosol in a manner similar to that required by diphtheria toxin. As shown for diphtheria toxin, the pH-dependent membrane translocation step of CNF1 could be mimicked at the level of the plasma membrane by a brief exposure to a pH of 相似文献   

High yield purification and first structural characterization of the full‐length bacterial toxin CNF1

The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full‐length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his‐tagged recombinant protein from E. coli extracts (CNF1‐H8). CNF1‐H8 was expressed at the low temperature of 15°C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1‐H8 functionality was confirmed by testing its activity on purified RhoA and on HEp‐2 cultured cells. Finally, a first structural characterization of the full‐length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:150–159, 2018     

Bidirectional effect of Wnt signaling antagonist DKK1 on the modulation of anthrax toxin uptake

LRP6,a co-receptor for the morphogen Wnt,aids endocytosis of anthrax complexes.Here we report that Dickkopf1(DKK1)protein,a secreted LRP6 ligand and antagonist,is also a modulator of anthrax toxin sensitivity.shRNA-mediated gene silencing or TALEN-mediated gene knockout of DKK1 reduced sensitivity of cells to PA-dependent hybrid toxins.However,unlike the solely inhibitory effect on Wnt signaling,the effects of DKK1 overexpression on anthrax toxicity were bidirectional,depending on its endogenous expression and cell context.Fluorescence microscopy and biochemical analyses showed that DKK1 facilitates internalization of anthrax toxins and their receptors,an event mediated by DKK1-LRP6-Kremen2 complex.Monoclonal antibodies against DKK1 provided dose-dependent protection to macrophages from killing by anthrax lethal toxin(LT).Our discovery that DKK1 forms ternary structure with LRP6 and Kremen2 in promoting PA-mediated toxin internalization provides a paradigm for bacterial exploitation of mechanisms that host cells use to internalize signaling proteins.     

Rho GTPase-activating bacterial toxins: from bacterial virulence regulation to eukaryotic cell biology

Studies on the interactions of bacterial pathogens with their host have provided an invaluable source of information on the major functions of eukaryotic and prokaryotic cell biology. In addition, this expanding field of research, known as cellular microbiology, has revealed fascinating examples of trans-kingdom functional interplay. Bacterial factors actually exploit eukaryotic cell machineries using refined molecular strategies to promote invasion and proliferation within their host. Here, we review a family of bacterial toxins that modulate their activity in eukaryotic cells by activating Rho GTPases and exploiting the ubiquitin/proteasome machineries. This family, found in human and animal pathogenic Gram-negative bacteria, encompasses the cytotoxic necrotizing factors (CNFs) from Escherichia coli and Yersinia species as well as dermonecrotic toxins from Bordetella species. We survey the genetics, biochemistry, molecular and cellular biology of these bacterial factors from the standpoint of the CNF1 toxin, the paradigm of Rho GTPase-activating toxins produced by urinary tract infections causing pathogenic Escherichia coli. Because it reveals important connections between bacterial invasion and the host inflammatory response, the mode of action of CNF1 and its related Rho GTPase-targetting toxins addresses major issues of basic and medical research and constitutes a privileged experimental model for host-pathogen interaction.     

Rho GTPases as targets of bacterial protein toxins

Several bacterial toxins target Rho GTPases, which constitute molecular switches in several signaling processes and master regulators of the actin cytoskeleton. The biological activities of Rho GTPases are blocked by C3-like transferases, which ADP-ribosylate Rho at Asn41, but not Rac or Cdc42. Large clostridial cytotoxins (e. g., Clostridium difficile toxin A and B) glucosylate Rho GTPases at Thr37 (Rho) or Thr35 (Rac/Cdc42), thereby inhibiting Rho functions by preventing effector coupling. The 'injected' toxins ExoS, YopE and SptP from Pseudomonas aeruginosa, Yersinia and Salmonella ssp., respectively, which are transferred into the eukaryotic target cells by the type-III secretion system, inhibit Rho functions by acting as Rho GAP proteins. Rho GTPases are activated by the cytotoxic necrotizing factors CNF1 and CNF2 from Escherichia coli and by the dermonecrotizing toxin DNT from B. bronchiseptica. These toxins deamidate/transglutaminate Gln63 of Rho to block the intrinsic and GAP-stimulated GTP hydrolysis, thereby constitutively activating the GTPases. Rho GTPases are also activated by SopE, a type-III system injected protein from Salmonella ssp., that acts as a GEF protein.     

The Rac-activating toxin cytotoxic necrotizing factor 1 oversees NK cell-mediated activity by regulating the actin/microtubule interplay

The cell cytoskeleton is widely acknowledged as a master for NK cell function. Specifically, actin filaments guide the NK cell binding to target cells, engendering the formation of the so-called immunological synapse, while microtubules direct the killer behavior. All these cytoskeleton-dependent activities are competently governed by the Rho GTPases, a family of regulatory molecules encompassing the three different subfamilies, Rho, Rac, and Cdc42. By using a Rac GTPase-activating bacterial protein toxin from Escherichia coli named cytotoxic necrotizing factor 1 (CNF1), we obtained results supporting the activation of Rac GTPase as a booster for effector cell-binding efficiency, recruitment ability, and, consequently, cytotoxicity. In particular, the augmented killer capacity of CNF1-treated NK cells was associated with the increased expression of certain cell adhesion or activation-associated molecules and the reshaping of the actin and microtubule networks. Importantly, CNF1 counteracted the activity exerted by toxins disrupting the cytoskeletal architecture. Hence, the activation of Rho GTPases, particularly Rac, induced by CNF1, appears to orchestrate a dynamic cross talk between microtubules and actin filaments, leading to a fruitful NK cell activity and polarization state. Our findings suggest that protein toxins might be viewed as modulators of NK cell cytotoxic activity and could possibly be regarded as useful pharmacological tools for certain Rho-linked immune diseases in the near future.     

Bacterial glycosyltransferase toxins

Mono‐glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide‐binding proteins of the Rho family. However, toxin‐induced glycosylation is not restricted to the Clostridia. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin‐catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.     

Molecular localization of the Escherichia coli cytotoxic necrotizing factor CNF1 cell-binding and catalytic domains

Cytotoxic necrotizing factor type 1 (CNF1) induces, in epithelial cells, the development of stress fibres via the GTPase Rho pathway. We showed that CNF1 is able to modify Rho both in vitro and in vivo. Recombinant N-terminal 33 kDa (CNF1Nter) and C-terminal 14.8–31.5 kDa (CNF1Cter) regions of the CNF1 protein allowed us to demonstrate that the N-terminal region contains the cell-binding domain of the toxin and that the C-terminal region is responsible for its catalytic activity. CNF1Nter lowered the activity of CNF1 when provided to cells before the toxin whereas CNF1Cter had no effect on CNF1 cell toxicity. CNF1Cter was sufficient to induce a typical CNF1 phenotype when microinjected into African green monkey kidney cells (Vero cells), and was able to modify Rho as previously reported for CNF1. The C-terminal domain lost its catalytic activity when deleted of various subdomains, suggesting a scattered distribution of catalytic-site amino acids. Elucidation of the CNF1 functional organization and analysis of amino acid homologies between CNFs (CNF1, CNF2), Pasteurella multocida toxin (PMT) and dermonecrotic toxin of Bordetella pertussis (DNT) allowed us to postulate that CNFs and DNT act on Rho via the same enzymatic activity located in their C-terminus, and that CNFs and PMT probably bind to analogous cell receptors.     

Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic Escherichia coli strains. Here, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as cellular receptor for CNF1 by co-precipitation of cell surface molecules with tagged toxin. The CNF1-Lu/BCAM interaction was verified by direct protein-protein interaction analysis and competition studies. These studies revealed amino acids 720 to 1014 of CNF1 as the binding site for Lu/BCAM. We suggest two cell interaction sites in CNF1: first the N-terminus, which binds to p37LRP as postulated before. Binding of CNF1 to p37LRP seems to be crucial for the toxin''s action. However, it is not sufficient for the binding of CNF1 to the cell surface. A region directly adjacent to the catalytic domain is a high affinity interaction site for Lu/BCAM. We found Lu/BCAM to be essential for the binding of CNF1 to cells. Cells deficient in Lu/BCAM but expressing p37LRP could not bind labeled CNF1. Therefore, we conclude that LRP and Lu/BCAM are both required for toxin action but with different functions.     

Cytotoxic necrotizing factor 1 fromEscherichia coli: a toxin with a new intracellular activity for eukaryotic cells

Certain pathogenicEscherichia coli strains elaborate a toxin, the cytotoxic necrotizing factor type 1 (CNF1). CNF1 covalently and specifically modifies the p21 Rho GTP-binding protein in mammalian cells by deamidation of the p21 Rho glutamine 63. CNF1 modification of Rho leads to permanent activation of the GTP-binding protein by blocking intrinsic and RhoGAP GTPase activities. Rho activation by CNF1 induces reorganization of the actin cytoskeleton into large stress fibers and the multiplication of focal contact points. Deamidation is a new catalytic activity described for an intracellularly acting toxin. Presented at the1st International Minisymposium on Cellular Microbiology: Cell Biology and Signalization in Host-Pathogen Interactions, Prague, October 6, 1997.     

Role of N-Terminal His6-Tags in Binding and Efficient Translocation of Polypeptides into Cells Using Anthrax Protective Antigen (PA)

It is of interest to define bacterial toxin biochemical properties to use them as molecular-syringe devices in order to deliver enzymatic activities into host cells. Binary toxins of the AB_7/8-type are among the most potent and specialized bacterial protein toxins. The B subunits oligomerize to form a pore that binds with high affinity host cell receptors and the enzymatic A subunit. This allows the endocytosis of the complex and subsequent injection of the A subunit into the cytosol of the host cells. Here we report that the addition of an N-terminal His₆-tag to different proteins increased their binding affinity to the protective antigen (PA) PA₆₃-channels, irrespective if they are related (C2I) or unrelated (gpJ, EDIN) to the AB_7/8-family of toxins. His₆-EDIN exhibited voltage-dependent increase of the stability constant for binding by a factor of about 25 when the trans-side corresponding to the cell interior was set to −70 mV. Surprisingly, the C. botulinum toxin C2II-channel did not share this feature of PA₆₃. Cell-based experiments demonstrated that addition of an N-terminal His₆-tag promoted also intoxication of endothelial cells by C2I or EDIN via PA₆₃. Our results revealed that addition of His₆-tags to several factors increase their binding properties to PA₆₃ and enhance the property to intoxicate cells.     

37-kDa laminin receptor precursor modulates cytotoxic necrotizing factor 1-mediated RhoA activation and bacterial uptake

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial toxin known to activate Rho GTPases and induce host cell cytoskeleton rearrangements. The constitutive activation of Rho GTPases by CNF1 is shown to enhance bacterial uptake in epithelial cells and human brain microvascular endothelial cells. However, it is unknown how exogenous CNF1 exhibits such phenotypes in eukaryotic cells. Here, we identified 37-kDa laminin receptor precursor (LRP) as the receptor for CNF1 from screening the cDNA library of human brain microvascular endothelial cells by the yeast two-hybrid system using the N-terminal domain of CNF1 as bait. CNF1-mediated RhoA activation and bacterial uptake were inhibited by exogenous LRP or LRP antisense oligodeoxynucleotides, whereas they were increased in LRP-overexpressing cells. These findings indicate that the CNF1 interaction with LRP is the initial step required for CNF1-mediated RhoA activation and bacterial uptake in eukaryotic cells.