Effects of Small Molecule Calcium-Activated Chloride Channel Inhibitors on Structure and Function of Accessory Cholera Enterotoxin (Ace) of Vibrio cholerae.

Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders.

Zonulin, iron status, and anemia in kidney transplant recipients: are they related?

BACKGROUND: In patients after kidney transplantation, anemia is relatively common and is associated with impaired kidney function, subclinical inflammatory state, and immunosuppressive treatment. Zonulin-prehaptoglobin-2, a newly discovered protein, is necessary for integrity of intracellular tight junctions in the gut. Taking into consideration iron metabolism, including its absorption in the gut, we designed a cross-sectional study to look for the possible interactions among zonulin, iron status, and anemia in kidney transplant recipients. METHODS: The study was performed on 72 stable kidney transplant recipients and 22 healthy volunteers. Zonulin, iron status, and inflammatory markers were assessed with the use of commercially available kits. RESULTS: Zonulin was significantly lower in kidney allograft recipients than in healthy volunteers (P < .001). Zonulin correlated with systolic blood pressure (r = -0.33; P < .05), thyroid-binding globulin (r = 0.24; P < .05), hematocrit (r = 0.28; P < .005), hemoglobin (r = 0.32; P < .01), total protein (r = -0.33; P < .01), erythrocyte count (r = 0.26; P < .05), and fasting glucose (r = -0.25; P < .05). Zonulin was not affected by sex, type of immunosuppressive therapy, presence of diabetes, coronary artery disease, heart failure, hypertension, or cause of end-stage renal disease. Zonulin was not related to any of the iron parameters studied. In multiple regression analysis, predictors of zonulin were total protein and thyroglobulin-binding protein, explaining 46% of variation. CONCLUSIONS: Zonulin, with its poorly defined function, does not seem to play a role in the anemia in kidney allograft recipients; however, it seems to be related to the absorption process in the gut.

RNA thermometer controls temperature-dependent virulence factor expression in Vibrio cholerae.

Vibrio cholerae is the bacterium that causes the diarrheal disease cholera. The bacteria experience a temperature shift as V. cholerae transition from contaminated water at lower temperatures into the 37 °C human intestine. Within the intestine, V. cholerae express cholera toxin (CT) and toxin-coregulated pilus (TCP), two main virulence factors required for disease. CT and TCP expression is controlled by the transcriptional activator protein ToxT. We identified an RNA thermometer motif in the 5' UTR of toxT, with a fourU anti-Shine-Dalgarno (SD) element that base pairs with the SD sequence to regulate ribosome access to the mRNA. RNA probing experiments demonstrated that the fourU element allowed access to the SD sequence at 37 °C but not at 20 °C. Moreover, mutations within the fourU element (U5C, U7C) that strengthened base-pairing between the anti-SD and SD sequences prevented access to the SD sequence even at 37 °C. Translation of ToxT-FLAG from the native toxT UTR was enhanced at 37 °C, compared with 25 °C in both Escherichia coli and V. cholerae. In contrast, the U5C, U7C UTR prevented translation of ToxT-FLAG even at 37 °C. V. cholerae mutants containing the U5C, U7C UTR variant were unable to colonize the infant mouse small intestine. Our results reveal a previously unknown regulatory mechanism consisting of an RNA thermometer that controls temperature-dependent translation of toxT, facilitating V. cholerae virulence at a relevant environmental condition found in the human intestine.

Structure of the cytoplasmic domain of TcpE, the inner membrane core protein required for assembly of the Vibrio cholerae toxin-coregulated pilus.

Type IV pili are long thin surface-displayed polymers of the pilin subunit that are present in a diverse group of bacteria. These multifunctional filaments are critical to virulence for pathogens such as Vibrio cholerae, which use them to form microcolonies and to secrete the colonization factor TcpF. The type IV pili are assembled from pilin subunits by a complex inner membrane machinery. The core component of the type IV pilus-assembly platform is an integral inner membrane protein belonging to the GspF superfamily of secretion proteins. These proteins somehow convert chemical energy from ATP hydrolysis by an assembly ATPase on the cytoplasmic side of the inner membrane to mechanical energy for extrusion of the growing pilus filament out of the inner membrane. Most GspF-family inner membrane core proteins are predicted to have N-terminal and central cytoplasmic domains, cyto1 and cyto2, and three transmembrane segments, TM1, TM2 and TM3. Cyto2 and TM3 represent an internal repeat of cyto1 and TM1. Here, the 1.88 Å resolution crystal structure of the cyto1 domain of V. cholerae TcpE, which is required for assembly of the toxin-coregulated pilus, is reported. This domain folds as a monomeric six-helix bundle with a positively charged membrane-interaction face at one end and a hydrophobic groove at the other end that may serve as a binding site for partner proteins in the pilus-assembly complex.

The 1.2 Å resolution crystal structure of TcpG, the Vibrio cholerae DsbA disulfide-forming protein required for pilus and cholera-toxin production.

The enzyme TcpG is a periplasmic protein produced by the Gram-negative pathogen Vibrio cholerae. TcpG is essential for the production of ToxR-regulated proteins, including virulence-factor pilus proteins and cholera toxin, and is therefore a target for the development of a new class of anti-virulence drugs. Here, the 1.2 Å resolution crystal structure of TcpG is reported using a cryocooled crystal. This structure is compared with a previous crystal structure determined at 2.1 Å resolution from data measured at room temperature. The new crystal structure is the first DsbA crystal structure to be solved at a sufficiently high resolution to allow the inclusion of refined H atoms in the model. The redox properties of TcpG are also reported, allowing comparison of its oxidoreductase activity with those of other DSB proteins. One of the defining features of the Escherichia coli DsbA enzyme is its destabilizing disulfide, and this is also present in TcpG. The data presented here provide new insights into the structure and redox properties of this enzyme, showing that the binding mode identified between E. coli DsbB and DsbA is likely to be conserved in TcpG and that the ß5-α7 loop near the proposed DsbB binding site is flexible, and suggesting that the tense oxidized conformation of TcpG may be the consequence of a short contact at the active site that is induced by disulfide formation and is relieved by reduction.

Zonulin, regulation of tight junctions, and autoimmune diseases.

Recent studies indicate that besides digestion and absorption of nutrients and water and electrolytes homeostasis, another key function of the intestine is to regulate the trafficking of environmental antigens across the host mucosal barrier. Intestinal tight junctions (TJs) create gradients for the optimal absorption and transport of nutrients and control the balance between tolerance and immunity to nonself antigens. To meet diverse physiological challenges, intestinal epithelial TJs must be modified rapidly and in a coordinated fashion by regulatory systems that orchestrate the state of assembly of the TJ multiprotein network. While considerable knowledge exists about TJ ultrastructure, relatively little is known about their physiological and pathophysiological regulation. Our discovery of zonulin, the only known physiologic modulator of intercellular TJs described so far, has increased our understanding of the intricate mechanisms that regulate the intestinal epithelial paracellular pathway and has led us to appreciate that its upregulation in genetically susceptible individuals leads to autoimmune diseases.

A unique role of the cholera toxin A1-DD adjuvant for long-term plasma and memory B cell development.

Adjuvants have traditionally been appreciated for their immunoenhancing effects, whereas their impact on immunological memory has largely been neglected. In this paper, we have compared three mechanistically distinct adjuvants: aluminum salts (Alum), Ribi (monophosphoryl lipid A), and the cholera toxin A1 fusion protein CTA1-DD. Their influence on long-term memory development was dramatically different. Whereas a single immunization i.p. with 4-hydroxy-3-nitrophenyl acetyl (NP)-chicken γ-globulin and adjuvant stimulated serum anti-NP IgG titers that were comparable at 5 wk, CTA1-DD-adjuvanted responses were maintained for >16 mo with a half-life of anti-NP IgG ∼36 wk, but <15 wk after Ribi or Alum. A CTA1-DD dose-dependent increase in germinal center (GC) size and numbers was found, with >60% of splenic B cell follicles hosting GC at an optimal CTA1-DD dose. Roughly 7% of these GC were NP specific. This GC-promoting effect correlated well with the persistence of long-term plasma cells in the bone marrow and memory B cells in the spleen. CTA1-DD also facilitated increased somatic hypermutation and affinity maturation of NP-specific IgG Abs in a dose-dependent fashion, hence arguing that large GC not only promotes higher Ab titers but also high-quality Ab production. Adoptive transfer of splenic CD80(+), but not CD80(-), B cells, at 1 y after immunization demonstrated functional long-term anti-NP IgG and IgM memory cells. To our knowledge, this is the first report to specifically compare and document that adjuvants can differ considerably in their support of long-term immune responses. Differential effects on the GC reaction appear to be the basis for these differences.

Mast cells contribute to the mucosal adjuvant effect of CTA1-DD after IgG-complex formation.

Mast cell activation is one of the most dramatic immune-mediated responses the body can encounter. In the worst scenario (i.e., anaphylaxis), this response is fatal. However, the importance of mast cells as initiators and effectors of both innate and adaptive immunity in healthy individuals has recently been appreciated. It was reported that mast cell activation can be used as an adjuvant to promote Ag-specific humoral immune responses upon vaccination. In this study, we have used a clinically relevant mucosal adjuvant, cholera toxin A1 subunit (CTA1)-DD, which is a fusion protein composed of CTA1, the ADP-ribosylating part of cholera toxin, and DD, two Ig-binding domains derived from Staphylococcus aureus protein A. CTA1-DD in combination with polyclonal IgG induced degranulation and production of TNF-alpha from mouse mast cells. Furthermore, CTA1-DD and polyclonal IgG complex induced mast cell degranulation in mouse skin tissue and nasal mucosa. We also found that intranasal immunization with hapten (4-hydroxy-3-nitrophenyl) acetyl (NP) coupled to chicken gammaglobulin admixed with CTA1-DD complexed with polyclonal IgG greatly enhanced serum IgG anti-NP Ab responses and stimulated higher numbers of NP-specific plasma cells in the bone marrow as compared with that observed in mice immunized with NP-chicken gammaglobulin with CTA1-DD alone. This CTA1-DD/IgG complex-mediated enhancement was mast cell dependent because it was absent in mast cell-deficient Kit(W-sh/W-sh) mice. In conclusion, our data suggest that a clinically relevant adjuvant, CTA1-DD, exerts additional augmenting effects through activation of mucosal mast cells, clearly demonstrating that mast cells could be further exploited for improving the efficacy of mucosal vaccines.

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery.

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.

Tight junctions as targets of infectious agents.

The epithelial barrier is a critical border that segregates luminal material from entering tissues. Essential components of this epithelial fence are physical intercellular structures termed tight junctions. These junctions use a variety of transmembrane proteins coupled with cytoplasmic adaptors, and the actin cytoskeleton, to attach adjacent cells together thereby forming intercellular seals. Breaching of this barrier has profound effects on human health and disease, as barrier deficiencies have been linked with the onset of inflammation, diarrhea generation and pathogenic effects. Although tight junctions efficiently restrict most microbes from penetrating into deeper tissues and contain the microbiota, some pathogens have developed specific strategies to alter or disrupt these structures as part of their pathogenesis, resulting in either pathogen penetration, or other consequences such as diarrhea. Understanding the strategies that microorganisms use to commandeer the functions of tight junctions is an active area of research in microbial pathogenesis. In this review we highlight and overview the tactics bacteria and viruses use to alter tight junctions during disease. Additionally, these studies have identified novel tight junction protein functions by using pathogens and their virulence factors as tools to study the cell biology of junctional structures.

A novel purification method for CNS projection neurons leads to the identification of brain vascular cells as a source of trophic support for corticospinal motor neurons.

One of the difficulties in studying cellular interactions in the CNS is the lack of effective methods to purify specific neuronal populations of interest. We report the development of a novel purification scheme, cholera toxin beta (CTB) immunopanning, in which a particular CNS neuron population is selectively labeled via retrograde axonal transport of the cell-surface epitope CTB, and then purified via immobilization with anti-CTB antibody. We have demonstrated the usefulness and versatility of this method by purifying both retinal ganglion cells and corticospinal motor neurons (CSMNs). Genomic expression analyses of purified CSMNs revealed that they express significant levels of many receptors for growth factors produced by brain endothelial cells; three of these factors, CXCL12, pleiotrophin, and IGF2 significantly enhanced purified CSMN survival, similar to previously characterized CSMN trophic factors BDNF and IGF1. In addition, endothelial cell conditioned medium significantly promoted CSMN neurite outgrowth. These findings demonstrate a useful method for the purification of several different types of CNS projection neurons, which in principle should work in many mammalian species, and provide evidence that endothelial-derived factors may represent an overlooked source of trophic support for neurons in the brain.

GM1 ganglioside contributes to retain the neuronal conduction and neuronal excitability in visceral and baroreceptor afferents.

GM1 ganglioside has a great impact on the function of nodes of Ranvier on myelinated fiber, suggesting its potential role to maintain the electrical and neuronal excitability of neurons. Here we first demonstrate that visceral afferent conduction velocity of myelinated and unmyelinated fibers are reduced significantly by tetrodotoxin (TTX) or cholera toxin-B subunits (CTX-B), and only the effects mediated by CTX-B are prevented by GM1 pre-treatment. At soma of myelinated A and unmyelinated C-type nodose ganglion neurons (NGNs), the action potential spike frequency reduced by CTX-B is also prevented by GM1. Additionally, the current density of both TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na(+) channels were significantly decreased by CTX-B without changing the voltage-dependent property. These data confirm that endogenous GM1 may play a dominant role in maintaining the electrical and neuronal excitability via modulation of sodium (Na(+)) channel around nodes and soma as well, especially TTX-S Na(+) channel, which is also confirmed by the reduction of spike amplitude and depolarization. Similar data are also extended to fluorescently identified and electrophysiologically characterized aortic baroreceptor neurons. These findings suggest that GM1 plays an important role in the neural modulation of electric and neuronal excitability in visceral afferent system.

Vibrio cholerae: cholera toxin.

The bacterial protein toxin of Vibrio cholerae, cholera toxin, is a major agent involved in severe diarrhoeal disease. Cholera toxin is a member of the AB toxin family and is composed of a catalytically active heterodimeric A-subunit linked with a homopentameric B-subunit. Upon binding to its receptor, GM0(1), cholera toxin is internalized and transported in a retrograde manner through the Golgi to the ER, where it is retrotranslocated to the cytosol. Here, cholera toxin reaches its intracellular target, the basolaterally located adenylate cyclase which becomes constitutively activated after toxin-induced mono-ADP-ribosylation of the regulating G(S)-protein. Elevated intracellular cAMP levels provoke loss of water and electrolytes which is manifested as the typical diarrhoea. The cholera toxin B-subunit displays the capacity to fortify immune responses to certain antigens, to act as a carrier and to be competent in inducing immunological tolerance. These unique features make cholera toxin a promising tool for immunologists.

Long ascending propriospinal projections from lumbosacral to upper cervical spinal cord in the rat.

The retrograde tracer cholera toxin beta-subunit (CTB) was used to trace long ascending propriospinal projections from neurons in the lumbosacral spinal cord to the upper cervical (C3) gray matter in adult male Sprague-Dawley rats. Following large 0.5 microl CTB injections restricted mainly to the upper cervical ventral horn (n=5), there were many lumbosacral CTB-positive neurons (14-17/section) in the intermediate gray and ventral horn (dorsal lamina VIII, medial VII extending into X) contralaterally, with fewer at corresponding ipsilateral locations. Labeled cells (4-8/section) were also observed in contralateral laminae IV-VI and the lateral spinal nucleus, with fewer ipsilaterally. Few labeled cells (<2/section) were observed in superficial laminae I-II. Smaller (0.15 microl) microinjections of CTB restricted to the upper cervical ventral gray matter labeled cells in contralateral laminae VII-VIII (approximately 6-9/section) with fewer ipsilaterally. There were relatively fewer (<2/section) in the intermediate dorsal horn and very few (<1/section) in lamina I. Larger (0.5 microl) CTB injections encompassing the C3 dorsal and ventral gray matter on one side labeled significantly more CTB-positive neurons (>6/section) in contralateral lamina I compared to ventral horn injections. These results suggest direct projections from ventromedially located neurons of lumbar and sacral segments to the contralateral ventral gray matter of upper cervical segments, as well as from neurons in the intermediate but not superficial dorsal horn. They further suggest that some lumbosacral superficial dorsal horn neurons project to the upper cervical dorsal horn. These propriospinal projections may be involved in coordinating head and neck movements during locomotion or stimulus-evoked motor responses.

Nanoscale glassification of gold substrates for surface plasmon resonance analysis of protein toxins with supported lipid membranes.

Surface plasmon resonance (SPR) spectroscopy, a powerful tool for biosensing and protein interaction analysis, is currently confined to gold substrates and the relevant surface chemistries involving dextran and functional thiols. Drawbacks of using self-assembled monolayers (SAMs) for SPR-related surface modification include limited stability, pinhole defects, bioincompatibility, and nonspecific protein adsorption. Here we report the development of stable nanometer-scale glass (silicate) layers on gold substrates for SPR analysis of protein toxins. The nanoscale silicate layers were built up with layer-by-layer deposition of poly(allylamine hydrochloride) and sodium silicate, followed by calcination at high temperature. The resulting silicate films have a thickness ranging from 2 to 15 nm and demonstrate outstanding stability in flow cell conditions. The use of these surfaces as a platform to construct supported bilayer membranes (SBMs) is demonstrated, and improved performance against protein adsorption on SBM-coated surfaces is quantified by SPR measurements. SBMs can be formed reproducibly on the silicate surface via vesicle fusion and quantitatively removed using injection of 5% Triton X-100 solution, generating a fresh surface for each test. Membrane properties such as lateral diffusion of the SBMs on the silicate films are characterized with photobleaching methods. Studies of protein binding with biotin/avidin and ganglioside/cholera toxin systems show detection limits lower than 1 microg/mL (i.e., nanomolar range), and the response reproducibility is better than 7% RSD. The method reported here allows many assay techniques developed for glass surfaces to be transferred to label-free SPR analysis without the need for adaptation of protocols and time-consuming synthetic development of thiol-based materials and opens new avenues for developing novel bioanalytical technologies for protein analysis.

Splenic marginal zone dendritic cells mediate the cholera toxin adjuvant effect: dependence on the ADP-ribosyltransferase activity of the holotoxin.

The in vivo mechanisms of action of most vaccine adjuvants are poorly understood. In this study, we present data in mice that reveal a series of critical interactions between the cholera toxin (CT) adjuvant and the dendritic cells (DC) of the splenic marginal zone (MZ) that lead to effective priming of an immune response. For the first time, we have followed adjuvant targeting of MZ DC in vivo. We used CT-conjugated OVA and found that the Ag selectively accumulated in MZ DC following i.v. injections. The uptake of Ag into DC was GM1 ganglioside receptor dependent and mediated by the B subunit of CT (CTB). The targeted MZ DC were quite unique in their phenotype: CD11c(+), CD8alpha(-), CD11b(-), B220(-), and expressing intermediate or low levels of MHC class II and DEC205. Whereas CTB only delivered the Ag to MZ DC, the ADP-ribosyltransferase activity of CT was required for the maturation and migration of DC to the T cell zone, where these cells distinctly up-regulated CD86, but not CD80. This interaction appeared to instruct Ag-specific CD4(+) T cells to move into the B cell follicle and strongly support germinal center formations. These events may explain why CT-conjugated Ag is substantially more immunogenic than Ag admixed with soluble CT and why CTB-conjugated Ag can tolerize immune responses when given orally or at other mucosal sites.

Fixation of Clostridium difficile toxin A and cholera toxin to intestinal brush border membranes from axenic and conventional mice.

We have tested the in vitro binding of Clostridium difficile toxin A (enterotoxin) and cholera toxin to intestinal brush border membranes prepared from either conventional or axenic mice. Membranes from axenic mice were shown to be saturated at a lower toxin A concentration (at least 2.5 times lower). Because there were no significant differences between membranes from axenic and conventional mice in binding at low toxin A concentrations, the presence of the normal microflora seems to increase the number but not the affinity of brush border membrane receptors on the enterocyte surface. Corroborating the in vitro results, we observed that conventional mice were more sensitive to the pathological effects of toxin A given intragastrically than were axenic mice. In contrast, there was no difference in the binding characteristics of cholera toxin between membranes from conventional and axenic mice. We conclude that the presence of the mouse intestinal bacteria increases the number of C. difficile toxin A intestinal receptors but does not influence cholera toxin receptors.

[Response to cholera toxin of 2 epithelial intestinal cell lines. Effect of Saccharomyces boulardii]. / Réponse à la toxine cholérique de deux lignées de cellules épithéliales intestinales. Effet de Saccharomyces boulardii.

Cholera toxin acts in vivo by activating intestinal adenylate cyclase. This study was designed to determine (1) whether normal rat epithelial intestinal cell lines (IRD 98 and IEC 17) respond to cholera toxin (CT) by an increased concentration of cyclic AMP and (2) whether the yeast Saccharomyces boulardii, which reduced CT-induced secretion of water and electrolytes using the isolated jejunal loop technique, has an effect on these models. The cAMP concentration evaluated in cells exposed to Saccharomyces boulardii and to cholera toxin (1 microgram/ml for 90 min) was compared to the concentration of cAMP obtained in control cells without yeast. Prior exposure of IRD 98 and IEC 17 cells to Saccharomyces boulardii, reduced CT-induced cAMP by 50 p. 100. This effect disappeared after destruction of the yeast by heating. Results show that the IRD 98 and IEC 17 cells are good models for in vitro investigation of the effects of cholera toxin. Our results suggests that Saccharomyces boulardii prevents the water and electrolyte secretion induced by cholera toxin.