Coli Cytotoxic Necrotizing Factor 1 Research Articles

Modeling and computational characterization of a Xanthomonas sp. Hypothetical protein identifies a remote ortholog of Burkholderia lethal factor 1

Burkholderia Lethal Factor 1 (BLF1) is a deamidase first characterized in Burkholderia pseudomallei. This enzyme inhibits cellular protein synthesis by deamidating a glutamine residue to a glutamic acid in its target protein, the eukaryotic translation initiation factor 4 A (eIF4A). In this work, we present the characterization of a hypothetical protein from Xanthomonas sp. Leaf131 as the first report of a BLF1 family ortholog outside of the Burkholderia genus. Although standard sequence similarity searches such as BLAST were not able to detect the homology between the Xanthomonas sp. Leaf131 hypothetical protein sequence and BLF1, our computed structure model for the Xanthomonas sp. hypothetical protein revealed structural similarities with an RMSD of 2.7 Å/164 Cα atoms and a TM-score of 0.72 when superposed. Structural comparisons of the Xanthomonas model structure against BLF1 and Escherichia coli cytotoxic necrotizing factor 1 (CNF1) revealed that the conserved signature LXGC motif and putative catalytic residues are structurally aligned thus signifying a level of functional or mechanistic similarity. Protein-protein docking analysis and molecular dynamics simulations also demonstrated that eIF4A could still be a possible target substrate for deamidation by XLF1 as it is for BLF1. We therefore propose that this Xanthomonas hypothetical protein be renamed as Xanthomonas Lethal Factor 1 (XLF1). Our work also provides further evidence of the utility of programs such as AlphaFold in bridging the computational function annotation transfer gap despite very low sequence identities of under 20%. Communicated by Ramaswamy H. Sarma

Escherichia coli Rho GTPase-activating toxin CNF1 mediates NLRP3 inflammasome activation via p21-activated kinases-1/2 during bacteraemia in mice.

SummaryInflammasomes are signaling platforms that are assembled in response to infection or sterile inflammation by cytosolic pattern recognition receptors (PRRs). The consequent inflammasome-triggered Caspase-1 activation is critical for the host defense against pathogens. During infection, NLRP3, a PRR also called Cryopyrin, triggers the assembly of an inflammasome activating Caspase-1 via the recruitment of ASC and Nek7. The NLRP3 inflammasome activation is tightly controlled both transcriptionally and post-translationally. Despite the importance of the NLRP3 inflammasome regulation in autoinflammatory and infectious diseases, little is known about the mechanism controlling the NLRP3 activation and the upstream signaling that regulates the NLRP3 inflammasome assembly. We have previously shown that the RhoGTPases-activating toxin from Escherichia coli, CNF1, activates Caspase-1, but the upstream mechanism is unclear. Here we provide evidence of the role of the NLRP3 inflammasome in sensing the activity of bacterial toxins and virulence factors that activate host RhoGTPases. We demonstrate that this activation relies on monitoring of the toxin’s activity on the RhoGTPase Rac2. We also show that the NLRP3 inflammasome is activated by a signaling cascade involving the P21 activated kinases (Pak) 1/2 and the Pak1-mediated phosphorylation of Threonine 659 of NLRP3, which is necessary for the NLRP3-Nek7 interaction, the inflammasome activation and the IL-1ß cytokine maturation. Furthermore, inhibition of the Pak-NLRP3 axis diminishes the bacterial clearance of CNF1-expressing UTI89 E. coli during bacteremia in mice. Altogether, our results establish Pak1/2 as critical regulators of the NLRP3 inflammasome and reveal the role of the Pak-NLRP3 signaling axis in vivo during bacteremia in mice.

The Bacterial Toxin CNF1 Protects Human Neuroblastoma SH-SY5Y Cells against 6-Hydroxydopamine-Induced Cell Damage: The Hypothesis of CNF1-Promoted Autophagy as an Antioxidant Strategy.

Several chronic neuroinflammatory diseases, including Parkinson’s disease (PD), have the so-called ‘redox imbalance’ in common, a dynamic system modulated by various factors. Among them, alteration of the mitochondrial functionality can cause overproduction of reactive oxygen species (ROS) with the consequent induction of oxidative DNA damage and apoptosis. Considering the failure of clinical trials with drugs that eliminate ROS directly, research currently focuses on approaches that counteract redox imbalance, thus restoring normal physiology in a neuroinflammatory condition. Herein, we used SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA), a neurotoxin broadly employed to generate experimental models of PD. Cells were pre-treated with the Rho-modulating Escherichia coli cytotoxic necrotizing factor 1 (CNF1), before the addition of 6-OHDA. Then, cell viability, mitochondrial morphology and dynamics, redox profile as well as autophagic markers expression were assessed. We found that CNF1 preserves cell viability and counteracts oxidative stress induced by 6-OHDA. These effects are accompanied by modulation of the mitochondrial network and an increase in macroautophagic markers. Our results confirm the Rho GTPases as suitable pharmacological targets to counteract neuroinflammatory diseases and evidence the potentiality of CNF1, whose beneficial effects on pathological animal models have been already proven to act against oxidative stress through an autophagic strategy.

The E. coli CNF1 as a Pioneering Therapy for the Central Nervous System Diseases

The Cytotoxic Necrotizing Factor 1 (CNF1), a protein toxin from pathogenic E. coli, modulates the Rho GTPases, thus, directing the organization of the actin cytoskeleton. In the nervous system, the Rho GTPases play a key role in several processes, controlling the morphogenesis of dendritic spines and synaptic plasticity in brain tissues. This review is focused on the peculiar property of CNF1 to enhance brain plasticity in in vivo animal models of central nervous system (CNS) diseases, and on its possible application in therapy.

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.

Prevention of Escherichia coli K1 penetration of the blood-brain barrier by counteracting the host cell receptor and signaling molecule involved in E. coli invasion of human brain microvascular endothelial cells.

Escherichia coli meningitis is an important cause of mortality and morbidity, and a key contributing factor is our incomplete understanding of the pathogenesis of E. coli meningitis. We have shown that E. coli penetration into the brain requires E. coli invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. E. coli invasion of HBMEC involves its interaction with HBMEC receptors, such as E. coli cytotoxic necrotizing factor 1 (CNF1) interaction with its receptor, the 67-kDa laminin receptor (67LR), and host signaling molecules including cytosolic phospholipase A(2)alpha (cPLA(2)alpha). In the present study, we showed that treatment with etoposide resulted in decreased expression of 67LR on HBMEC and inhibited E. coli invasion of HBMEC. Pharmacological inhibition of cysteinyl leukotrienes, lipoxygenated products of arachidonic acid released by cPLA(2)alpha, using montelukast (an antagonist of the type 1 cysteinyl leukotriene receptor) also inhibited E. coli invasion of HBMEC. E. coli penetration into the brain was significantly decreased by etoposide as well as by montelukast, and a combination of etoposide and montelukast was significantly more effective in inhibiting E. coli K1 invasion of HBMEC than single agents alone. These findings demonstrate for the first time that counteracting the HBMEC receptor and signaling molecule involved in E. coli invasion of HBMEC provides a novel approach for prevention of E. coli penetration into the brain, the essential step required for development of E. coli meningitis.

Autoantibodies from pemphigus patients cause skin blistering by inhibition of Rho GTPases

The autoimmune blistering skin diseases pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are mediated by autoantibodies (IgG) against the desmosomal cadherins desmoglein 1 (Dsg 1) and Dsg 3. Recently we demonstrated that PF IgG do not reduce Dsg 1-mediated adhesion by steric hindrance but rather required cell-dependent mechanisms (J. Clin. Invest. 2005, Oct 6 Epub). Here we provide evidence that PV-IgG (directed to Dsg 1 and Dsg 3) and PF-IgG (directed to Dsg 1) induce skin blistering by inhibition of Rho family GTPases. PV-IgG and PF-IgG caused epidermal blistering in biopsies of human skin and intercellular gap formation in cultured keratinocytes. Moreover, autoantibodies reduced Dsg 1-mediated binding assessed by laser tweezers and inactivated cellular Rho family GTPases. Both PV-IgG and PF-IgG induced inactivation of Rho A whereas PV-IgG additionally inhibited Rac 1 and Cdc42. Concomitant activation of these GTPases by E. coli cytotoxic necrotizing factor 1 (CNF-1) outbalanced autoantibody-induced Rho-GTPase inactivation and largely abolished antibody-induced effects on human epidermis and cultured keratinocytes. These data indicate that inactivation of Rho-GTPases is important for pemphigus pathogenesis. DFG SFB487

Inhibition of vessel permeability by TNP-470 and its polymer conjugate, caplostatin

Angiogenesis inhibitors, such as TNP-470 and the nontoxic HPMA copolymer-TNP-470 (caplostatin), are emerging as a class of anticancer drugs. We report that TNP-470 and caplostatin inhibit vascular hyperpermeability of tumor blood vessels as well as that induced in mouse skin by different mediators. Treatment with TNP-470 or angiostatin for 3 days was sufficient to reduce permeability of tumor blood vessels, delayed-type hypersensitivity, and pulmonary edema induced by IL-2. TNP-470 also inhibited VPF/VEGF-induced phosphorylation of VEGFR-2, calcium influx, and RhoA activation in endothelial cells. These results identify an activity of TNP-470, that of inhibiting vessel hyperpermeability. This activity likely contributes to TNP-470's antiangiogenic effect and suggests that caplostatin can be used in the treatment of cancer and inflammation.

Identification and characterization of the Pasteurella multocida toxin translocation domain.

The Pasteurella multocida toxin (PMT) is a potent mitogen which enters the cytosol of eukaryotic cells via a low pH membrane translocation event. In common with the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), the core of the PMT translocation domain is composed of two predicted hydrophobic helices (H1 - residues 402-423, H2 - 437-457) linked by a hydrophilic loop (PMT-TL - 424-436). The peptide loop contains three acidic residues (D425, D431 and E434), which may play a role equivalent to D373, D379 and E382/383 in CNF1. To test this hypothesis, a series of point mutants was generated in which acidic residues were mutated into the permanently charged positive residue lysine. Individual mutation of D425, D431 and E434 each caused a four- to sixfold reduction in toxin activity. Interestingly, mutation of D401 located immediately outside the predicted helix-loop-helix motif completely abolished toxin activity. Individual mutations did not affect cell binding nor greatly altered toxin structure, but did prevent translocation of the surface-bound proteins into the cytosol after a low pH pulse. Moreover, we demonstrate using an in vitro assay that PMT undergoes a pH-dependent membrane insertion.

Constitutive activation of Rho proteins by CNF-1 influences tight junction structure and epithelial barrier function.

The apical-most epithelial intercellular junction, referred to as the tight junction (TJ), regulates paracellular solute flux in diverse physiological and pathological states. TJ affiliations with the apical filamentous actin (F-actin) cytoskeleton are crucial in regulating TJ function. F-actin organization is influenced by the Rho GTPase family, which also controls TJ function. To explore the role of Rho GTPases in regulating TJ structure and function, we utilized Escherichia coli cytotoxic necrotizing factor-1 (CNF-1) as a tool to activate constitutively Rho, Rac and Cdc42 signaling in T84 polarized intestinal epithelial monolayers. The biological effects of the toxin were polarized to the basolateral membrane, and included profound reductions in TJ gate function, accompanied by displacement of the TJ proteins occludin and zonula occludens-1 (ZO-1), and reorganization of junction adhesion molecule-1 (JAM-1) away from the TJ membrane. Immunogold electron microscopy revealed occludin and caveolin-1 internalization in endosomal/caveolar-like structures in CNF-treated cells. Immunofluorescence/confocal microscopy suggested that a pool of internalized occludin went to caveolae, early endosomes and recycling endosomes, but not to late endosomes. This provides a novel mechanism potentially allowing occludin to evade a degradative pathway, perhaps allowing efficient recycling back to the TJ membrane. In contrast to the TJ, the characteristic ring structure of proteins in adherens junctions (AJs) was largely preserved despite CNF-1 treatment. CNF-1 also induced displacement of a TJ-associated pool of phosphorylated myosin light chain (p-MLC), which is normally also linked to the F-actin contractile machinery in epithelial cells. The apical perjunctional F-actin ring itself was maintained even after toxin exposure, but there was a striking effacement of microvillous F-actin and its binding protein, villin, from the same plane. However, basal F-actin stress fibers became prominent and cabled following basolateral CNF-1 treatment, and the focal adhesion protein paxillin was tyrosine phosphorylated. This indicates differences in Rho GTPase-mediated control of distinct F-actin pools in polarized cells. Functionally, CNF-1 profoundly impaired TJ/AJ assembly in calcium switch assays. Re-localization of occludin but not E-cadherin along the lateral membrane during junctional reassembly was severely impaired by the toxin. A balance between activity and quiescence of Rho GTPases appears crucial for both the generation and maintenance of optimal epithelial barrier function. Overactivation of Rho, Rac and Cdc42 with CNF-1 seems to mirror key barrier-function disruptions previously reported for inactivation of RhoA.

Activation of rho GTPases by cytotoxic necrotizing factor 1 induces macropinocytosis and scavenging activity in epithelial cells.

Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function. It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors. In this case, the internalized material recycles back to the cell surface. We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes. In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation. Taken together, these findings indicate that CNF1-induced "switching on" of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes. The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages.

Identification of the Region of Rho Involved in Substrate Recognition by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) and the Bordetella dermonecrotic toxin (DNT) activate Rho GTPases by deamidation of Gln(63) of RhoA (Gln(61) of Cdc42 and Rac). In addition, both toxins possess in vitro transglutaminase activity in the presence of primary amines. Here we characterized the region of Rho essential for substrate recognition by the toxins using Rho/Ras chimeras as protein substrates. The chimeric protein Ras55Rho was deamidated or transglutaminated by CNF1. Rat pheochromocytoma PC12 cells microinjected with Ras55Rho developed formation of neurite-like structures after treatment with the CNF1 holotoxin indicating activation of the Ha-Ras chimera and Ras-like effects in intact cells. The Ras59Rho78Ras chimera protein contained the minimal Rho sequence allowing deamidation or transglutamination by CNF1. A peptide covering mainly the switch II region and consisting of amino acid residues Asp(59) through Asp(78) of RhoA was substrate for CNF1. Changes of amino acid residues Arg(68) or Leu(72) of RhoA into the corresponding residues of Ras (R68ARhoA and L72QRhoA) inhibited deamidation and transglutamination of the mutants by CNF1. In contrast to CNF1, DNT did not modify Rho/Ras chimeras or the switch II peptide (Asp(59) through Asp(78)). Glucosylation of RhoA at Thr(37) blocked deamidation by DNT but not by CNF. The data indicate that CNF1 recognizes Rho GTPases exclusively in the switch II region, whereas the substrate recognition by DNT is characterized by additional structural requirements.

The 5' region of cnf1 harbours a translational regulatory mechanism for CNF1 synthesis and encodes the cell-binding domain of the toxin.

The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) is organized into three functional domains: the N-terminal part containing the cell-binding domain, a putative central membrane-spanning region, and a C-terminal catalytic region. On the basis of competition assays between CNF1 and GST-recombinant proteins containing different N-terminal fragments, and point mutations, we restricted the binding region to the first 190 amino acids. Hydrophilic amino acids 53-75 are strictly necessary to cell receptor recognition. Using different cnf1-lacZ translational fusions, we demonstrated that the mRNA corresponding to the first 48 codons of cnf1 is involved in the translational regulation of CNF1 synthesis. This regulation consists of both a positive and a negative control. The positive control is exerted by codons 6-20, including a putative downstream box that enhances the translational expression of cnf1. The negative control depends on codons 45-48. In this region, an anti-Shine-Dalgarno sequence, highly homologous to the core of the internal complementary sequence already reported for growth rate-regulated metabolic genes, has been detected. To some extent, the inner structural organization of CNF1 would thus suggest the compiling of several functions in a single mRNA protein system.

Effect of Escherichia coli cytotoxic necrotizing factor 1 on repair of human bladder cell monolayers in vitro.

We hypothesized that Escherichia coli cytotoxic necrotizing factor 1 (CNF1) might impair migration or proliferation of bladder cells and could potentially interfere with repair of the bladder epithelium. Using experimentally wounded human T24 bladder epithelial cell monolayers as an in vitro model, we found that both the number of T24 cells and the maximum distance they migrated into wounded regions was significantly decreased by bacterial extracts containing E. coli CNF1.

Effects of cytotoxic necrotizing factor 1 and lethal toxin on actin cytoskeleton and VE-cadherin localization in human endothelial cell monolayers.

Integrity of the vascular endothelium is largely dependent on endothelial cell shape and establishment of intercellular junctions. Certain pathogenic bacterial toxins alter the cytoskeletal architecture of intoxicated cells by modulating the GTPase activity of p21 Rho family proteins. In the present study we have analyzed the effect of Rho-directed toxins on the actin cytoskeleton and monolayer integrity of endothelial cells. We report here that Escherichia coli cytotoxic necrotizing factor 1 (CNF1) activates Rho in human umbilical vein endothelial cells (HUVEC). In confluent monolayers, CNF1 treatment induces prominent stress fiber formation without significantly modifying peripheral localization of VE-cadherin, a specific marker of vascular endothelial cell adherens junctions. Further, Rho activation with CNF1 blocks thrombin-induced redistribution of VE-cadherin staining and gap formation in HUVEC monolayers. Inhibition of Rho by prolonged treatment of cells with C3 exoenzyme (Clostridium botulinum) eliminates actin stress fibers without disrupting the continuity of VE-cadherin staining, indicating that Rho-dependent stress fibers are not required for maintaining this adhesion receptor at sites of intercellular contact. Lethal toxin (Clostridium sordellii), an inhibitor of Rac as well as Ras and Rap, potently disrupts the actin microfilament system and monolayer integrity in HUVEC cultures.

Deamidation of Cdc42 and Rac by Escherichia coli cytotoxic necrotizing factor 1: activation of c-Jun N-terminal kinase in HeLa cells.

Recently, Escherichia coli cytotoxic necrotizing factor 1 (CNF1) was shown to activate the low-molecular-mass GTPase RhoA by deamidation of Gln63, thereby inhibiting intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities (G. Schmidt, P. Sehr, M. Wilm, J. Selzer, M. Mann, and K. Aktories, Nature 387:725-729, 1997; G. Flatau, E. Lemichez, M. Gauthier, P. Chardin, S. Paris, C. Fiorentini, and P. Boquet, Nature 387:729-733, 1997). Here we report that in addition to RhoA, Cdc42 and Rac also are targets for CNF1 in vitro and in intact cells. Treatment of HeLa cells with CNF1 induced a transient formation of microspikes and formation of membrane ruffles. CNF1 caused a transient 10- to 50-fold increase in the activity of the c-Jun N-terminal kinase. Tryptic peptides of Cdc42 obtained from CNF1-treated cells by immunoprecipitation exhibited an increase in mass of 1 Da compared to control peptides, indicating the deamidation of glutamine 61 by the toxin. The same increase in mass was observed with the respective peptides obtained from CNF1-modified recombinant Cdc42 and Rac1. Modification of recombinant Cdc42 and Rac1 by CNF1 inhibited intrinsic and GAP-stimulated GTPase activities and retarded binding of 2'(3')-O-(N-methylanthraniloyl)GDP. The data suggest that recombinant as well as cellular Cdc42 and Rac are substrates for CNF1.

Intestinal epithelial cytoskeleton selectively constrains lumen-to-tissue migration of neutrophils.

Migration of neutrophils (polymorphonuclear leukocytes; PMN) across polarized epithelia is asymmetrical: basolateral-to-apical (physiologically directed) migration is far more efficient than migration in the reverse direction, suggesting the presence of luminal retention signal(s). Following pilot observations, we used polarized intestinal epithelial monolayers (T84) to examine whether asymmetrical constraint of migration afforded by the epithelial cytoskeleton might underlie such retention signals. Rearrangement of epithelial cortical F-actin accompanied PMN transepithelial migration (in either direction) and was prevented by preloading monolayers with the F-actin stabilizing agent phallacidin. Although phallacidin preloading did not influence physiologically directed PMN transepithelial migration, such treatment greatly enhanced migration in the reverse direction (i.e., effective loss of luminal retention signal). 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) preloading also prevented epithelial cortical actin rearrangements and selectively resulted in loss of luminal retention signal(s). BAPTA preloading did not influence resistance or forskolin-induced Cl- secretion, and phallacidin preloading did not influence resistance or carbachol-induced Cl- secretion, suggesting that barrier function and surface polarity were maintained under these conditions. These and supplementary data suggest that epithelial actin (but not microtubule) cytoskeletal reordering asymmetrically influences PMN migration and underlies, at least in part, the observed signal that biases for retention of PMN in the luminal space.

E. Coli cytotoxic necrotizing factor 1 (CNF1) and its effects on cells: C. Fiorentini, 1L. Falzano, 1G. Donelli, 1E. Oswald, 2M. R. Popoff3 and P. Boquet3 ( 1Department of Ultrastructures, Istituto Superiore di Sanita, Rome, Italy; 2Laboratoire de génie de l'hygiène et des procédés alimentaires, Institut National de la Recherche Agronomique, Massy, France; and 3 Unité des Toxines Microbiennes, Institut

E. Coli cytotoxic necrotizing factor 1 (CNF1) and its effects on cells: C. Fiorentini, 1L. Falzano, 1G. Donelli, 1E. Oswald, 2M. R. Popoff3 and P. Boquet3 ( 1Department of Ultrastructures, Istituto Superiore di Sanita, Rome, Italy; 2Laboratoire de génie de l'hygiène et des procédés alimentaires, Institut National de la Recherche Agronomique, Massy, France; and 3 Unité des Toxines Microbiennes, Institut