Clostridium difficile toxins A and B directly stimulate human mast cells

Clostridium difficile toxins A and B (TcdA and TcdB) are the causative agents of antibiotic-associated pseudomembranous colitis. Mucosal mast cells play a crucial role in the inflammatory processes underlying this disease. We studied the direct effects of TcdA and TcdB on the human mast cell line HMC-1 with respect to degranulation, cytokine release, and the activation of proinflammatory signal pathways. TcdA and TcdB inactivate Rho GTPases, the master regulators of the actin cytoskeleton. The inactivation of Rho GTPases induced a reorganization of the actin cytoskeleton accompanied by morphological changes of cells. The TcdB-induced reorganization of the actin cytoskeleton in HMC-1 cells reduced the number of electron-dense mast cell-specific granules. Accordingly, TcdB induced the release of hexosaminidase, a marker for degranulation, in HMC-1 cells. The actin rearrangement was found to be responsible for degranulation since latrunculin B induced a comparable hexosaminidase release. In addition, TcdB as well as latrunculin B induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 and also resulted in a p38 MAPK-dependent increased formation of prostaglandins D(2) and E(2). The autocrine stimulation of HMC-1 cells by prostaglandins partially contributed to the degranulation. Interestingly, TcdB-treated HMC-1 cells, but not latrunculin B-treated HMC-1 cells, showed a strong p38 MAPK-dependent increase in interleukin-8 release. Differences in the mast cell responses to TcdB and latrunculin B are probably due to the presence of functionally inactive Rho GTPases in toxin-treated cells. Thus, the HMC-1 cell line is a promising model for studying the direct effects of C. difficile toxins on mast cells independently of the tissue context.     

Inhibition of protein kinase C attenuates Pseudomonas aeruginosa elastase-induced epithelial barrier disruption

Pseudomonas aeruginosa pulmonary infection compromises the human airway epithelium, and can be especially devastating to immunocompromised or debilitated individuals. We have reported earlier that P. aeruginosa elastase (PE) increases paracellular permeability in epithelial cell monolayers by mechanisms involving tight junction (TJ) disruption and cytoskeletal reorganization, leading to destruction of epithelial barrier function. The aim of this study was to investigate putative TJ targets and potential mechanisms by which PE induces barrier disruption. We found that PE decreased localization of TJ proteins, occludin and zonula occludens (ZO)-1, in membrane fractions, and induced reorganization of F-actin within 1 hour. Although inhibition of protein kinase (PK) C α/β signaling modestly altered the extent of cytoskeletal disruption and ZO-1 translocation, we found PKC signaling to play a significant role in decreased occludin functionality during PE exposure. Furthermore, elevated PKC levels correlated with decreased levels of TJ proteins in membrane fractions, and increased paracellular permeability in a time-dependent manner. Therefore, we conclude that PKC signaling is involved during PE-induced epithelial barrier disruption via TJ translocation and cytoskeletal reorganization. Specifically, occludin, as well as associated ZO-1 and F-actin, may be early targets of PE pathogenesis occurring via a PKC-dependent pathway.     

Neutrophil Transmigration in Inflammatory Bowel Disease Is Associated with Differential Expression of Epithelial Intercellular Junction Proteins

Inflammatory bowel disease (IBD) consisting of ulcerative colitis (UC) and Crohn's (CD) typically displays a waxing and waning course punctuated by disease flares that are characterized by transepithelial migration of neutrophils (PMN) and altered barrier function. Since epithelial barrier function is primarily regulated by the apical most intercellular junction referred to as the tight junction (TJ), our aim was to examine expression of TJ and adherens junction (AJ) proteins in relation to PMN infiltration in mucosal tissue samples from patients with active IBD. Expression of epithelial intercellular TJ proteins (occludin, ZO-1, claudin-1, and JAM) and subjacent AJ (beta-catenin and E-cadherin) proteins were examined by immunoflourescence/confocal microscopy, immunohistochemistry, and Western blotting. Colonic mucosa from patients with UC revealed dramatic, global down-regulation of the key TJ transmembrane protein occludin in regions of actively transmigrating PMN and in quiescent areas in the biopsy samples. Significant decreases in occludin expression were observed at the protein and mRNA levels by Western and Northern blotting. In contrast, expression of other TJ and AJ proteins such as ZO-1, claudin-1, JAM, beta-catenin, and E-cadherin were down-regulated only in epithelial cells immediately adjacent to transmigrating PMN. Analysis of inflamed mucosa from Crohn's disease patients mirrored the results obtained with UC patients. No change in TJ and AJ protein expression was observed in colonic epithelium from patients with collagenous colitis or lymphocytic colitis that are respectively characterized by a thickened subepithelial collagen plate and increased intraepithelial lymphocytes. These results suggest that occludin expression is diminished in IBD by mechanisms distinct from those regulating expression of other intercellular junction proteins. We speculate that down-regulation of epithelial occludin may play a role in enhanced paracellular permeability and PMN transmigration that is observed in active inflammatory bowel disease.     

Bacterial protein toxins inhibiting low-molecular-mass GTP-binding proteins

The Rho GTPases, which belong to the Ras superfamily of low-molecular-mass GTP-binding proteins, are the preferred intracellular targets of bacterial protein toxins. The Rho GTPases RhoA/B/C, Rac1/2 and Cdc42 are the master regulators of the actin cytoskeleton. Clostridium difficile toxins A and B, the causative agents of the antibiotic-associated pseudomembranous colitis, are intracellularly acting cytotoxins which mono-glucosylate the Rho GTPases. Clostridium botulinum C3 toxin, which is not related to the clostridial neurotoxins, catalyses ADP-ribosylation of RhoA/B/C but not of other Rho GTPases. Glucosylation as well as ADP-ribosylation result in functional inactivation of Rho causing disassembly of the actin cytoskeleton.     

EspF Interacts with nucleation-promoting factors to recruit junctional proteins into pedestals for pedestal maturation and disruption of paracellular permeability

Many pathogenic bacteria subvert normal host cell processes by delivering effector proteins which mimic eukaryotic functions directly into target cells. EspF is a multifunctional protein injected into host cells by attaching and effacing pathogens, but its mechanism of action is not understood completely. In silico analyses of EspF revealed two key motifs: proline-rich domains and PDZ domain binding motifs. Such functional domains may allow EspF to act as an actin nucleation-promoting factor by mimicking host proteins. In agreement with these predictions, we found that EspF from rabbit enteropathogenic Escherichia coli (E22) participates in the regulation of actin polymerization by binding to a complex of proteins at the tight junctions (TJ). EspF bound to actin and profilin throughout the course of infection. However, after 2 h of infection, EspF also bound to the neural Wiskott-Aldrich syndrome protein and to the Arp2/3, zonula occludens-1 (ZO-1), and ZO-2 proteins. Moreover, EspF caused occludin, claudin, ZO-1, and ZO-2 redistribution and loss of transepithelial electrical resistance, suggesting that actin sequestration by EspF may cause local actin depolymerization leading to EspF-induced TJ disruption. Furthermore, EspF caused recruitment of these TJ proteins into the pedestals. An E22 strain lacking EspF did not cause TJ disruption and pedestals were smaller than those induced by the wild-type strain. Additionally, the pedestals were located mainly in the TJ. The overexpression of EspF caused bigger pedestals located along the length of the cells. Thus, actin sequestration by EspF allows the recruitment of junctional proteins into the pedestals, leading to the maturation of actin pedestals and the disruption of paracellular permeability.     

Involvement of Ras-related Rho proteins in the mechanisms of action of Clostridium difficile toxin A and toxin B.

Toxins A and B of Clostridium difficile are responsible for pseudomembranous colitis, a disease that afflicts a substantial number of hospitalized patients treated with antibiotics. A major effect of these proteins is the disruption of the actin cytoskeleton. Recently, I. Just, G. Fritz, K. Aktories, M. Giry, M. R. Popoff, P. Boquet, S. Hegenbarth, and C. von Eichel-Streiber (J. Biol. Chem. 269:10706-10712, 1994) implicated Rho proteins as cellular targets of C. difficile toxin B, since pretreatment of cells or purified Rho with toxin prevented subsequent ADP-ribosylation of Rho by exoenzyme C3. Moreover, they showed that overexpression of Rho proteins in cells suppressed cell rounding normally associated with exposure of cells to C. difficile toxin B. Here we expand these findings by showing directly that Rho proteins are covalently modified by both C. difficile toxins A and B. In addition, we demonstrate that the stability of toxin-modified Rho in NIH 3T3 cells is dramatically reduced. Finally, we show that C. difficile toxins A and B do not have similar effects on the closely related Rac and CDC42 GTP-binding proteins.     

Clostridium difficile toxins: mechanism of action and role in disease

As the leading cause of hospital-acquired diarrhea, Clostridium difficile colonizes the large bowel of patients undergoing antibiotic therapy and produces two toxins, which cause notable disease pathologies. These two toxins, TcdA and TcdB, are encoded on a pathogenicity locus along with negative and positive regulators of their expression. Following expression and release from the bacterium, TcdA and TcdB translocate to the cytosol of target cells and inactivate small GTP-binding proteins, which include Rho, Rac, and Cdc42. Inactivation of these substrates occurs through monoglucosylation of a single reactive threonine, which lies within the effector-binding loop and coordinates a divalent cation critical to binding GTP. By glucosylating small GTPases, TcdA and TcdB cause actin condensation and cell rounding, which is followed by death of the cell. TcdA elicits effects primarily within the intestinal epithelium, while TcdB has a broader cell tropism. Important advances in the study of these toxins have been made in the past 15 years, and these are detailed in this review. The domains, subdomains, and residues of these toxins important for receptor binding and enzymatic activity have been elegantly studied and are highlighted herein. Furthermore, there have been major advances in defining the role of these toxins in modulating the inflammatory events involving the disruption of cell junctions, neuronal activation, cytokine production, and infiltration by polymorphonuclear cells. Collectively, the present review provides a comprehensive update on TcdA and TcdB's mechanism of action as well as the role of these toxins in disease.     

Cytoskeletal modulation and tyrosine phosphorylation of tight junction proteins are associated with mainstream cigarette smoke-induced permeability of airway epithelium

Cigarette smoke increases the permeability of the lung epithelium. Consequences of increased permeability include increased access of toxins and pathogens from the air spaces to the interstitium and even the blood stream, and leakage of fluids into the air spaces. The mechanisms for permeability alterations have not been elucidated for airway epithelia. By analogy with other types of epithelia, we hypothesized that changes in the phosphorylation status and function of tight junction (TJ) or cytoskeletal proteins might mediate the smoke-induced permeability changes. We investigated the effects of exposure to mainstream cigarette smoke (MS) on cultures of Calu-3 cells, an airway epithelial cell line. Specifically, MS exposure caused increases in phosphorylation of the myosin-binding subunit (MBS) of myosin phosphatase and myosin light chain (MLC), proteins involved in the regulation of actin polymerization. These results implicate activation of Rho kinase (ROCK), consistent with previously reported data indicating that inhibition of ROCK activation suppressed MS-induced increases in permeability. MS exposure also increased polymerized (filamentous) actin (f-actin) content and caused redistribution of the TJ proteins from the normal apical circumferential band to a more basal location. The translocation of the TJ proteins was spatially associated with local increases in both f-actin and macromolecular permeability. Finally, MS exposure increased tyrosine phosphorylation of occludin but not ZO-1 and decreased association between the two TJ proteins. These results indicate that MS exposure causes alterations in cytoskeletal and TJ structure and function, resulting in increased macromolecular permeability that may contribute to the adverse health effects of MS.     

Cytosolic delivery and characterization of the TcdB glucosylating domain by using a heterologous protein fusion

TcdB from Clostridium difficile glucosylates small GTPases (Rho, Rac, and Cdc42) and is an important virulence factor in the human disease pseudomembranous colitis. In these experiments, in-frame genetic fusions between the genes for the 255 amino-terminal residues of anthrax toxin lethal factor (LFn) and the TcdB(1-556) coding region were constructed, expressed, and purified from Escherichia coli. LFnTcdB(1-556) was enzymatically active and glucosylated recombinant RhoA, Rac, Cdc42, and substrates from cell extracts. LFnTcdB(1-556) plus anthrax toxin protective antigen intoxicated cultured mammalian cells and caused actin reorganization and mouse lethality, all similar to those caused by wild-type TcdB.     

Evidence for holin function of tcdE gene in the pathogenicity of Clostridium difficile.

Toxigenic strains of Clostridium difficile produce two large bacterial toxins called toxins A (TcdA) and B (TcdB). tcdA and tcdB genes are located on the pathogenicity locus of C. difficile, a unique characteristic of toxigenic strains of this species. Intergenic to the two toxin genes is tcdE, a small 501-bp open reading frame of unknown function. Expression of the tcdE gene in Escherichia coli caused bacterial cell death. Computational analysis of the amino acid sequence of TcdE revealed structural features that are strikingly similar to a class of bacteriophage proteins called holins. Holins are cytolytic proteins that cause lysis of bacterial hosts to effect the release of progeny phages. Further analysis of the recombinant clone expressing TcdE by transmission electron microscopy confirmed that the site of action of TcdE is on the bacterial cell membrane. The results provide evidence that TcdE is structurally and functionally similar to holin proteins. TcdE may function as a lytic protein to facilitate the release of TcdA and TcdB to the extracellular environment, as these toxins lack signal peptide.     

Apically exposed, tight junction-associated beta1-integrins allow binding and YopE-mediated perturbation of epithelial barriers by wild-type Yersinia bacteria

Using polarized epithelial cells, primarily MDCK-1, we assessed the mode of binding and effects on epithelial cell structure and permeability of Yersinia pseudotuberculosis yadA-deficient mutants. Initially, all bacteria except the invasin-deficient (inv) mutant adhered apically to the tight junction areas. These contact points of adjacent cells displayed beta1-integrins together with tight junction-associated ZO-1 and occludin proteins. Indeed, beta1-integrin expression was maximal in the tight junction area and then gradually decreased along the basolateral membranes. Wild-type bacteria also opened gradually the tight junction to paracellular permeation of different-sized markers, viz., 20-, 40-, and 70-kDa dextrans and 45-kDa ovalbumin, as well as to their own translocation between adjacent cells in intimate contact with beta1-integrins. The effects on the epithelial cells and their barrier properties could primarily be attributed to expression of the Yersinia outer membrane protein YopE, as the yopE mutant bound but caused no cytotoxicity. Moreover, the apical structure of filamentous actin (F-actin) was disturbed and tight junction-associated proteins (ZO-1 and occludin) were dispersed along the basolateral membranes. It is concluded that the Yersinia bacteria attach to beta1-integrins at tight junctions. Via this localized injection of YopE, they perturb the F-actin structure and distribution of proteins forming and regulating tight junctions. Thereby they promote paracellular translocation of bacteria and soluble compounds.     

Role of stretch on tight junction structure in alveolar epithelial cells.

Previous studies have demonstrated that high tidal volumes can cause interstitial and alveolar edema, with degradation of pulmonary epithelial barrier integrity. Separate studies have shown that F-actin disruption and decreased intracellular ATP (ATP(i)) levels in the nonpulmonary epithelium can increase tight junction (TJ) permeability. We hypothesized that large epithelial stretch perturbs ATP(i) and actin architecture, each of which adversely affects TJ structure, and thus increases TJ permeability. Primary alveolar epithelial cells were subjected to a uniform 25% or 37% change in surface area (DeltaSA), cyclic biaxial stretch (15 cycles/min) for 1 h, or treated with either glycolytic metabolic inhibitors or cytoskeletal disrupting agents. Unstretched, untreated cells served as controls. Changes in the TJ proteins occludin and ZO-1 were determined by immunocytochemical evaluation. A stretch amplitude of 25% DeltaSA did not produce any significant cytologic changes compared with controls, but an amplitude of 37% DeltaSA stretch resulted in significant decreases in the intensity of the peripheral occludin band, the degree of cell-cell attachment (CCA), and total cellular occludin content. ATP depletion significantly diminished the occludin band intensity and decreased CCA. Actin disruption did not affect TJ protein band intensities (although the occludin distribution became punctate) but altered CCA. Untreated cells stretched cyclically at 25% or 50% DeltaSA for 1 h had significantly decreased ATP(i) compared with unstretched controls. These results suggest that stretch-induced ATP(i) reduction and actin perturbation disrupt TJ structure and CCA, which may lead to the alveolar flooding associated with high tidal volumes.     

Enteropathogenic Escherichia coli changes distribution of occludin and ZO-1 in tight junction membrane microdomains in vivo

Diarrhea is a disease caused by enteropathogenic Escherichia coli (EPEC) infection, which caused the deaths of several hundred thousand children each year. However, the molecular mechanisms underlying EPEC infection in vivo are not fully understood. In the present study, we used the C57BL/6J mouse as an in vivo model of EPEC infection and investigated the effect of EPEC on tight junction (TJ) structure and barrier function. TJ ultrastructure was studied by transmission electron microscopy and a small molecule tracer biotin was used to examine the paracellular permeability of the colon. The distribution of TJ proteins occludin and ZO-1 in the epithelium was investigated by immunofluorescence microscopy. Our results demonstrated that TJ structure was disrupted following EPEC infection. And the morphological changes of TJ were accompanied by increased paracellular permeability which led to impairment of TJ barrier function. Immunofluorescency analysis revealed that occludin and ZO-1 were translocated from villous membrane to the cytoplasm in intestinal epithelial cells during EPEC invasion. Moreover, wild-type EPEC and the mutant EPEC strain, ΔespF, had similar effects on barrier function and TJ protein localization at 5 days postinfection. Our findings demonstrate that EPEC infection in vivo led to disruption of tight junction barrier function. These results may provide insights into the molecular mechanism of the pathogenesis of EPEC infection.     

n-3 polyunsaturated fatty acids prevent disruption of epithelial barrier function induced by proinflammatory cytokines

Defects in tight junction barrier have been considered as an important etiologic factor of Crohn's disease. n-3 polyunsaturated fatty acids (PUFAs) exert beneficial effects on inflammatory bowel disorders. However, the mechanisms remain unknown. We found that docosahexaenoic acid (DHA, 22:6 n-3) and eicosapentaenoic acid (EPA, 20:5 n-3) changed lipid environment in membrane microdomains of tight junction in vitro. n-3 PUFAs treatment effectively prevented the redistribution of occludin and ZO-1 and distortion of TJ morphology, reduced transepithelial electrical resistance induced by IFN-gamma and TNF-alpha. We also observed dramatic reorganization of TJ proteins in epithelial lateral membrane following treatment with these cytokines. Our findings for first time indicate that n-3 PUFAs play an important role in proinflammatory cytokines-induced permeability defects and epithelial barrier dysfunction by modifying lipid environment in membrane microdomains of tight junction.     

Escherichia coli Heat-Stable Toxin b Impairs Intestinal Epithelial Barrier Function by Altering Tight Junction Proteins

Escherichia coli heat-stable toxin b (STb) causes diarrhea in animals. STb binds to sulfatide, its receptor, and is then internalized. In the cytoplasm, through a cascade of events, STb triggers the opening of ion channels, allowing ion secretion and water loss and leading to diarrhea. Tight junctions (TJs) are well known for controlling paracellular traffic of ions and water by forming a physical intercellular barrier in epithelial cells, and some bacterial toxins are known to affect adversely TJs. The present study aimed at determining the effect of STb on TJs. T84 cells were treated for 24 h with purified STb and a nontoxic STb mutant (D30V). Transepithelial resistance (TER), paracellular flux marker, and confocal microscopy were used to analyze the effect of STb on TJs. Purified STb caused a significant reduction of TER parallel to an increase in paracellular permeability compared to the results seen in untreated cells or mutant D30V. The increased paracellular permeability was associated with a marked alteration of F-actin stress fibers. F-actin filament dissolution and condensation were accompanied by redistribution and/or fragmentation of ZO-1, claudin-1, and occludin. These changes were also observed following treatment of T84 cells with an 8-amino-acid peptide found in the STb sequence corresponding to a consensus sequence of Vibrio cholerae Zot toxin. These effects were not observed with a scrambled peptide or mutant D30V. Our findings indicate that STb induces epithelial barrier dysfunction through changes in TJ proteins that could contribute to diarrhea.     

Hypoxia-induced cytoskeleton disruption in alveolar epithelial cells

Alveolar hypoxia, a common feature of many respiratory disorders, has been previously reported to induce functional changes, particularly a decrease of transepithelial Na and fluid transport. In polarized epithelia, cytoskeleton plays a regulatory role in transcellular and paracellular transport of ions and fluid. We hypothesized that exposure to hypoxia could damage cytoskeleton organization, which in turn, may adversely affect ion and fluid transport. Primary rat alveolar epithelial cells (AEC) were exposed to either mild (3% O(2)) or severe (0.5% O(2)) hypoxia for 18 h or to normoxia (21% O(2)). First, mild and severe hypoxia induced a disorganization of actin, a major protein of the cytoskeleton, reflected by disruption of F-actin filaments. Second, alpha-spectrin, an apical cytoskeleton protein, which binds to actin cytoskeleton and Na transport proteins, was cleaved by hypoxia. Pretreatment of AEC by a caspase inhibitor (z-VAD-fmk; 90 microM) blunted hypoxia-induced spectrin cleavage as well as hypoxia-induced decrease in surface membrane alpha-ENaC and concomitantly induced a partial recovery of hypoxia-induced decrease of amiloride-sensitive Na transport at 3% O(2). Finally, tight junctions (TJs) proteins, which are linked to actin and are a determinant of paracellular permeability, were altered by mild and severe hypoxia: hypoxia induced a mislocalization of occludin from the TJ to cytoplasm and a decrease in zonula occludens-1 protein level. These modifications were associated with modest changes in paracellular permeability at 0.5% O(2,) as assessed by small 4-kD dextran flux and transepithelial resistance measurements. Together, these findings indicate that hypoxia disrupted cytoskeleton and TJ organization in AEC and may participate, at least in part, to hypoxia-induced decrease in Na transport.     

Activation of transient receptor potential vanilloid subtype 1 increases expression and permeability of tight junction in normal and hyposecretory submandibular gland

Tight junction (TJ) is an important structure that regulates material transport through the paracellular pathway across the epithelium, but its significance in salivary physiology and pathogenesis of salivary dysfunctional diseases is not fully understood. We previously demonstrated that a functional transient receptor potential vanilloid subtype 1 (TRPV1) expresses in submandibular gland (SMG). However, association of TRPV1-induced saliva secretion with TJ remains unknown. Here we explored the effect of TRPV1 activation on expression and function of TJ of rabbit SMG in vitro and in vivo. RT-PCR and western blot analysis revealed that capsaicin upregulated expression of zonula occludin-1 (ZO-1), claudin (Cldn)-3, and -11, but not Cldn-1, -2, -4, -5, and -7 in cultured SMG cells. Capsaicin also increased the entering of 4?kDa FITC-dextran into the acinar lumen, induced redistribution of cytoskeleton F-actin under confocal microscope, and these effects were abolished by preincubation of capsazepine, a TRPV1 antagonist, indicating that activation of TRPV1 increases expression and permeability of TJ in SMG. Additionally, in a hyposecretory model induced by rabbit SMG transplantation, the expression of ZO-1, Cldn-3, and -11 was decreased, whereas other TJs remained unaltered. The structure of TJ was impaired and the width of apical TJs was reduced under transmission electron microscope, concomitant with diminished immunofluorescence of F-actin in peri-apicolateral region, indicating impaired TJ expression and decreased paracellular permeability in the transplanted SMG. Moreover, topical capsaicin cream increased secretion, decreased TJ structural injury, reversed TJ expression levels, and protected F-actin morphology from disarrangement in transplanted SMGs. These data provide the first evidence to demonstrate that TJ components, particularly ZO-1, Cldn-3, and -11 have important roles in secretion of SMG under both physiological and pathophysiological conditions. The injury in TJ integrity was involved in the hypofunctional SMGs, and TRPV1 might be a potential target to improve saliva secretion through modulating expression and function of TJs.     

A chimeric toxin vaccine protects against primary and recurrent Clostridium difficile infection

The global emergence of Clostridium difficile infection (CDI) has contributed to the recent surge in severe antibiotic-associated diarrhea and colonic inflammation. C. difficile produces two homologous glucosylating exotoxins, TcdA and TcdB, both of which are pathogenic and require neutralization to prevent disease occurrence. However, because of their large size and complex multifunctional domain structures, it has been a challenge to produce native recombinant toxins that may serve as vaccine candidates. Here, we describe a novel chimeric toxin vaccine that retains major neutralizing epitopes from both toxins and confers complete protection against primary and recurrent CDI in mice. Using a nonpathogenic Bacillus megaterium expression system, we generated glucosyltransferase-deficient holotoxins and demonstrated their loss of toxicity. The atoxic holotoxins induced potent antitoxin neutralizing antibodies showing little cross-immunogenicity or protection between TcdA and TcdB. To facilitate simultaneous protection against both toxins, we generated an active clostridial toxin chimera by switching the receptor binding domain of TcdB with that of TcdA. The toxin chimera was fully cytotoxic and showed potent proinflammatory activities. This toxicity was essentially abolished in a glucosyltransferase-deficient toxin chimera, cTxAB. Parenteral immunization of mice or hamsters with cTxAB induced rapid and potent neutralizing antibodies against both toxins. Complete and long-lasting disease protection was conferred by cTxAB vaccinations against both laboratory and hypervirulent C. difficile strains. Finally, prophylactic cTxAB vaccination prevented spore-induced disease relapse, which constitutes one of the most significant clinical issues in CDI. Thus, the rational design of recombinant chimeric toxins provides a novel approach for protecting individuals at high risk of developing CDI.     

Oestradiol decreases colonic permeability through oestrogen receptor β-mediated up-regulation of occludin and junctional adhesion molecule-A in epithelial cells

Oestradiol modulates paracellular permeability and tight junction (TJ) function in endothelia and reproductive tissues, but whether the ovarian hormones and cycle affect the paracellular pathway in the intestinal epithelium remains unclear. Oestrogen receptors (ERs) are expressed in intestinal epithelial cells, and oestradiol regulates epithelium formation. We examined the effects of oestrous cycle stage, oestradiol benzoate (EB), and progesterone (P) on colonic paracellular permeability (CPP) in the female rat, and whether EB affects expression of the TJ proteins in the rat colon and the human colon cell line Caco-2. In cyclic rats, CPP was determined through lumen-to-blood ⁵¹Cr-labelled EDTA clearance, and in Ussing chambers for dextran permeability. CPP was also examined in ovariectomized (OVX) rats treated with P or EB, with and without the ER antagonist ICI 182,780, or with the selective agonists for ERα (propyl pyrazole triol; PPT) or ERβ (diarylpropionitrile; DPN). In oestrus rats, CPP was reduced ( P < 0.01) relative to dioestrus. In OVX rats, EB dose-dependently decreased CPP, an effect mimicked by DPN and blocked by ICI 182,780, whereas P had no effect. Oestradiol increased occludin mRNA and protein in the colon ( P < 0.05), but not zona occludens (ZO)-1. Further, EB and DPN enhanced occludin and junctional adhesion molecule (JAM)-A expression in Caco-2 cells without change in ZO-1, an effect blocked by ICI 182,780. These data show that oestrogen reinforces intestinal epithelial barrier through ERβ-mediated up-regulation of the transmembrane proteins occludin and JAM-A determining paracellular spaces. These findings highlight the importance of the ERβ pathway in the control of colonic paracellular transport and mucosal homeostasis.     

Difference in the cytotoxic effects of toxin B from Clostridium difficile strain VPI 10463 and toxin B from variant Clostridium difficile strain 1470

Glucosylation of RhoA, Rac1, and Cdc42 by Clostridium difficile toxin B from strain VPI 10463 (TcdB) results in actin reorganization (cytopathic effect) and apoptosis (cytotoxic effect). Toxin B from variant C. difficile strain 1470 serotype F (TcdBF) differs from TcdB with regard to substrate proteins, as it glucosylates Rac1 and R-Ras but not RhoA and Cdc42. In this study, we addressed the question of whether the cellular effects of the toxins depend on their protein substrate specificity. Rat basophilic leukemia (RBL) cells were synchronized using the thymidine double-block technique. We show that cells were most sensitive to the cytotoxic effect of TcdB in S phase, as analyzed in terms of phosphatidyl serine externalization, fragmentation of nuclei, and activation of caspase-3; in contrast, TcdBF induced only a marginal cytotoxic effect, suggesting that inactivation of RhoA (but not of Rac1) was required for the cytotoxic effect. The glucosylation of Rac1 was correlated to the cytopathic effect of either toxin, suggesting a close connection of the two effects. The cytotoxic effect of TcdB was executed by caspase-3, as it was responsive to inhibition by acetyl-Asp-Met-Gln-Asp-aldehyde (Ac-DMQD-CHO), an inhibitor of caspase-3. The viability of TcdB-treated RBL cells was reduced, whereas the viability of TcdBF-treated cells was unchanged, further confirming that inactivation of RhoA is required for the cytotoxic effect. In conclusion, the protein substrate specificity of the glucosylating toxins determines their biological activity.