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Results for “toxin secretion system”

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PubMed1993

Cerulenin inhibits the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in brefeldin A-resistant cell lines.

We have found that cerulenin, an antibiotic that inhibits de novo fatty acid and cholesterol biosynthesis and fatty acylation of proteins, strongly inhibited the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in a brefeldin A (BFA)-resistant mutant of Vero cells (BER-40). The protective effect of cerulenin against ricin was also observed in two other BFA-resistant cell lines, Madin-Darby canine kidney, and PtK1 cells. In contrast to BER-40 cells, no significant effect of cerulenin was observed in Vero cells. Cerulenin did not affect the binding of ricin to the cell-surface receptors, but reduced significantly the internalization of ricin in BER-40 cells; no effect of cerulenin on the binding or internalization of ricin was observed in Vero, PtK1, and Madin-Darby canine kidney cells. Endocytic uptake of fluid-phase markers such as horseradish peroxidase and lucifer yellow was inhibited by cerulenin in BER-40 cells, but the endocytosis of transferrin via the coated pit/coated vesicle pathway was slightly increased. Cerulenin inhibited the degradation and excretion of ricin in BER-40 cells, and this effect of cerulenin was not observed in Vero cells. Furthermore, cerulenin inhibited the bulk protein secretion in a dose-dependent manner, with BER-40 cells being more susceptible than Vero cells. These results suggest that in addition to its effect on endocytosis, cerulenin interferes with the intracellular trafficking or processing of toxin molecules, and the vesicle transport system in BER-40 cells appears to be cerulenin-sensitive. Since addition of fatty acids and cholesterol did not reverse the effects of cerulenin, the protective effect of cerulenin against protein toxins is not due to an inhibition of de novo fatty acids and cholesterol biosynthesis.

ADP Ribose Transferases↗

PubMed1998

Enteric bacterial pathogens, villus atrophy and microbial growth.

The effects of three bacterial pathogens on the villus architecture of small intestines and the role that bacterial virulence factors play in pathogenesis are described. Bacterial pathogens cause a spectrum of effects ranging from severe tissue damage to a lack of perceptible damage. Enterotoxigenic Escherichia coli, which cause acute and severe diarrhea, does so by producing potent toxins, but these toxins act by altering the biological activity in epithelial cells. However, the cells are not damaged. Enteropathogenic E. coli and Salmonella, on the other hand cause various degrees of tissue damage. As part of their pathogenesis, they employ a type III protein secretion system to orchestrate internal changes in target cells. The expression of many virulence related genes is tightly regulated and appears to be turned on in response to cues found in the intestinal tract. The consequences of this level of regulation also is discussed.

Animals↗

PubMed2003

Bordetella type III secretion induces caspase 1-independent necrosis.

The Bordetella bronchiseptica type III (TIII) secretion system induces cytotoxicity in infected macrophages and epithelial cells. In this report we characterize the cell death phenotype and compare it to the TIII-dependent cytotoxicity induced by Yersinia enterocolitica and Shigella flexneri. Bordetella bronchiseptica strain RB58 was able to induce cell death in J774A.1 macrophages with the same efficiency as Shigella and Yersinia, but only B. bronchiseptica was able to kill epithelial cells in a TIII-dependent manner. Primary macrophages from caspase 1-/- mice were susceptible to RB58-mediated killing, suggesting that unlike Shigella and Salmonella, caspase 1 does not mediate cell death. RB58-induced cytotoxicity was not inhibited by addition of the pan-caspase inhibitor zVAD, and Western blot analyses of RB58-infected HeLa cells indicated that neither caspase 3 nor 7 was cleaved and PARP remained in its full-length active form. Morphologically the RB58-infected HeLa cells resembled necrotic rather than apoptotic cells, exhibiting cytoplasmic swelling and extensive membrane blebbing in the absence of nuclear changes. The addition of exogenous glycine, which has been shown to prevent necrotic cell death by blocking non-specific ion fluxes across the plasma membrane, blocked RB58-induced cytotoxicity. Addition of cyclosporin A which prevents the opening of the mitochondrial permeability pore, had no effect on RB58-infected cells. We conclude that the B. bronchiseptica TIII secretion system induces a mode of cell death consistent with necrosis that is distinct from that of Yersinia and Shigella.

Animals↗

PubMed2002

Gene expression, mutation, and structure-function relationship of scorpion toxin BmP05 active on SK(Ca) channels.

Four peptide inhibitors of small-conductance Ca(2+)-activated, apamin-sensitive K(+) channels (SK(Ca)) have been isolated from the venom of the Chinese scorpion Buthus martensi, named BmP01, BmP02, BmP03, and BmP05, respectively [Romi-Lebrun, R. (1997) Eur. J. Biochem. 245, 457-464]. Among them BmP05 with 31 amino acid residues has been intensively studied due to its most potent toxicity. To investigate the structure-function relationship of BmP05, its wild type and seven mutants (their C-termini unamidated) were successfully expressed in the yeast secretion system and purified with a high yield over 8 mg/L. Their toxicity to mice and electrophysiological activity on the K(+) currents (SK(Ca) and Kv) in rat adrenal chromaffin cells were measured and compared. The results indicated the following: (1) As a selective antagonist against SK(Ca), 1 microM rBmP05 is equivalent to 0.2 microM apamin, and its IC(50) is 0.92 microM. (2) The basic residues Lys and Arg located at positions 6 and 13 in the N-terminal alpha-helix region are essential and synergetic in the interaction of the toxin with SK(Ca). (3) Disruption of the alpha-helix by mutation of Gln at position 9 with Pro results in almost total loss of toxicity. (4) The C-terminal residue His31 plays an auxiliary role in the interaction of the toxin with SK(Ca). (5) The beta-turn connecting two beta-sheets near the C-terminal part is responsible for the specificity of the toxin to the different subtypes of K(+) channels.

Amino Acid Sequence↗

PubMed2005

Determinants of natriuretic peptide gene expression.

The cardiac natriuretic peptides (NP) atrial natriuretic factor or peptide (ANF or ANP) and brain natriuretic peptide (BNP) are polypeptide hormones synthesized, stored and secreted mainly by cardiac muscle cells (cardiocytes) of the atria of the heart. Both ANF and BNP are co-stored in storage granules referred to as specific atrial granules. The biological properties of NP include modulation of intrinsic renal mechanisms, the sympathetic nervous system, the rennin-angiotensin-aldosterone system (RAAS) and other determinants, of fluid volume, vascular tone and renal function. Studies on the control of baseline and stimulated ANF synthesis and secretion indicate at least two types of regulated secretory processes in atrial cardiocytes: one is stretch-stimulated and pertussis toxin (PTX) sensitive and the other is Gq-mediated and is PTX insensitive. Baseline ANF secretion is also PTX insensitive. In vivo, it is conceivable that the first process mediates stimulated ANF secretion brought about by changes in central venous return and subsequent atrial muscle stretch as observed in acute extracellular fluid volume expansion. The second type of stimulation is brought about by sustained hemodynamic and neuroendocrine stimuli such as those observed in congestive heart failure.

Amino Acid Sequence↗

PubMed2006

Virulence genotyping of Salmonella spp. with multiplex PCR.

The purpose of this study was to develop a multiplex polymerase chain reaction (PCR) protocol useful in the virulence genotyping of Salmonella spp. with the idea that genotyping could augment current Salmonella characterization and typing methods. Seventeen genes associated with Salmonella invasion, fimbrial production, toxin production, iron transport, and intramacrophage survival were targeted by three PCR reactions. Most of these genes are required for full Salmonella virulence in a murine model, and many are also located on Salmonella pathogenicity islands (PAIs) and are associated with type III secretion systems (TTSSs). Once the success of procedures that used positive and negative control strains was verified, the genotypes of 78 Salmonella isolates incriminated in avian salmonellosis (primarily from sick, commercially reared chickens and turkeys) and 80 Salmonella isolates from apparently healthy chickens or turkeys were compared. Eleven of the 17 genes tested (invA, orgA, prgH, tolC, spaN [invJ], sipB, sitC, pagC, msgA, spiA, and iroN) were found in all of the isolates. Another (sopB) was present in all isolates from sick birds and all but one isolate from healthy birds. The remaining five genes (lpfC, cdtB, sifA, pefA, and spvB) were found in 10%-90% of the isolates from sick birds and 3.75%-90% of the healthy birds. No significant differences in the occurrence of these genes between the two groups of isolates were detected. These results suggest that these virulence genes, and presumably the PAls and TTSSs with which they are associated, are widely distributed among Salmonella isolates of birds, regardless of whether their hosts of origin have been identified as having salmonellosis.

Animals↗

PubMed1984

[Neuro-hormonal regulation of the function of the enterocyte].

The intestinal epithelial cell, or enterocyte, performs a dual function: absorption and secretion. Absorption takes place in the villi and secretion in the crypts (and perhaps also in the villi). Both functions are controlled by complex extra- and intracellular systems. The extracellular system is mediated by neuromediators released in the subcellular space. Some mediators, like the vasoactive intestinal peptide (VIP) or the prostaglandins, stimulate secretion while others, like enkephalins, stimulate absorption. All these mediators act on intracellular metabolic processes by activating "second messengers" (e.g. amp or intracellular calcium) which in turn regulate the enterocyte transport system. Diarrheogenic processes are frequently due to disturbances in enterocyte control with abnormal stimulation of the secretory processes. Some pancreatic cancers and sympathicoblastomas release VIP into the blood stream in sufficient amounts to stimulate cAMP production by the enterocytes and, consequently, secretion. The cholera toxin and the E-coli thermolabile toxin can also induce stimulation of intracellular cAMP production. Conversely, the most promising antidiarrhoeal drugs are antisecretory agents, like opiates, which stimulate the enterocyte absorption processes.

Animals↗

PubMed1991

Distribution, persistence, and recall of serum and salivary antibody responses to peroral immunization with protein antigen I/II of Streptococcus mutans coupled to the cholera toxin B subunit.

After peroral immunization of mice with surface protein antigen (Ag) I/II of Streptococcus mutans conjugated to the cholera toxin B (CTB) subunit, cells actively secreting immunoglobulin A (IgA) antibodies specific for Ag I/II, but not for CT, were induced in the salivary glands; salivary IgA anti-Ag I/II antibodies and total salivary IgA were also elevated. The development of large numbers of IgA and IgG antibody-secreting cells in the mesenteric lymph nodes and spleen and high levels of serum IgA and IgG antibodies to Ag I/II and CT demonstrated that a response to both antigens occurred. At least two to three intragastric doses of 15 micrograms or more of Ag I/II-CTB conjugate, plus free CT as an adjuvant, were needed to induce the salivary IgA anti-Ag I/II response, which peaked at about 35 days and persisted at lower levels for 5 to 6 months. A single booster intragastric immunization did not induce enhanced salivary IgA anti-Ag I/II antibodies relative to the primary response, but serum IgA and IgG antibodies to both Ag I/II and CT showed evidence of marked anamnestic responses. The results indicated that relatively long-term mucosal IgA antibody responses could be induced by peroral immunization with small quantities of a CTB-conjugated protein. However, additional factors governed the distribution of cells secreting antibodies of different specificities, or capable of mounting anamnestic responses, between different compartments of the mucosal and circulatory immune systems.

Adjuvants, Immunologic↗

PubMed1987

Prostaglandin inhibition of acid is cAMP dependent.

Prostaglandin E (PGE) potently inhibits acid secretion stimulated by histamine, but not by acetylcholine or gastrin, and is accompanied by decreased intracellular cAMP. Adenylate cyclase receptor systems are composed of three complex proteins: cell receptor, nucleotide binding protein, and the catalytic subunit. The exact mechanism of PGE interaction with this complex remains unclear and elucidation of this site of action is the purpose of this study. We utilized molecular probes directed at the various components of adenylate cyclase. Cholera toxin alters the stimulatory subunit of the nucleotide binding proteins (Ns), rendering it resistant to normal deactivation, whereas N-ethylmaleimide (NEM) blocks the inhibitory subunit (Ni). Forskolin acts as a direct activator of the catalytic subunit of adenylate cyclase and 8-bromo-cAMP acts as a cyclic AMP mimetic. We measured in vitro acid secretion in isolated parietal cells by the assessment of [14C]aminopyrine (AP) accumulation. The PGE1 analog (miso) and the PGE2 analog (DMPG) were incubated in graded doses (10(-11) to 10(-6) M) with histamine (10(-6) M). Miso (10(-7) M) reduced AP accumulation to 21 +/- 8% of histamine alone (100%) and DMPG (10(-6) M) reduced AP to 61 +/- 9% (P less than 0.005 for both). AP accumulation stimulated by 8-Br-cAMP (10(-6) M) and forskolin (10(-6) M) was not significantly affected by either PGE analog (P greater than 0.05) suggesting that the site of PGE interaction is proximal to the activation of the catalytic subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1983

5-Hydroxytryptamine and cholera secretion: a histochemical and physiological study in cats.

The effect of cholera toxin on the content of 5-hydroxytryptamine (5-HT) in the enterochromaffin cells of the cat small intestine was estimated by cytofluorimetry of individual enterochromaffin cells at varying times after exposing the intestinal mucosa to the toxin. The observed changes in 5-HT levels in the enterochromaffin cells were correlated with the simultaneously measured rate of net fluid transport across the intestinal epithelium. Intestinal segments exposed to cholera toxin showed a statistically significant decrease in 5-HT levels of enterochromaffin cells compared with segments exposed to heat-inactivated cholera toxin. A good correlation (r = 0.73) was found between relative 5-HT fluorescence in enterochromaffin cells and net fluid transport across the intestinal epithelium. Thus, a diminished 5-HT content was associated with a decreased rate of fluid absorption or an increased rate of secretion. A hypothesis is presented for explaining the possible role of the enterochromaffin cells in the pathophysiology of cholera secretion.

Animals↗

PubMed2005

Kinetics of local and systemic immune responses after vaginal immunization with recombinant cholera toxin B subunit in humans.

Vaginal vaccination seems to be the best strategy for inducing specific immunoglobulin A (IgA) and IgG antibody responses in the female genital tract. The relative efficiencies of one, two, and three vaginal doses of recombinant cholera toxin B subunit (CTB) in generating mucosal and systemic immune responses in healthy women were evaluated, and the kinetics of the immune responses were monitored for responding volunteers for up to 12 months after the last vaccination. A single dose of CTB failed to generate CTB-specific IgA antibody responses in cervical secretions. Two vaccinations induced significant increases in IgA antitoxin titers in seven of nine volunteers, and four volunteers also developed IgG antitoxin responses. The magnitudes of the responses were 20-fold for IgA antitoxin and 7.1-fold for IgG antitoxin. A third vaccination did not significantly increase the antitoxin responses, although the frequency of IgG responses was slightly higher than that after the second vaccination. In serum, CTB-specific antibodies were observed already after a single vaccination. However, two vaccinations were required to induce marked IgA as well as IgG antitoxin titer increases in the majority of volunteers. The postvaccination levels of antitoxin antibodies in serum were comparable after two and three vaccinations. At 12 months after vaccination, significantly elevated IgA and IgG antitoxin levels in cervical secretions could still be detected in approximately half of the volunteers who had initially responded to the vaccine. Antitoxin titer increases in serum were found in most of the vaccinees at follow-up.

Adult↗

PubMed2005

Heteropentameric cholera toxin B subunit chimeric molecules genetically fused to a vaccine antigen induce systemic and mucosal immune responses: a potential new strategy to target recombinant vaccine antigens to mucosal immune systems.

Noninvasive mucosal vaccines are attractive alternatives to parenteral vaccines. Although the conjugation of vaccine antigens with the B subunit of cholera toxin (CTB) is one of the most promising strategies for vaccine delivery to mucosal immune systems, the molecule cannot tolerate large-protein fusion, as it severely impairs pentamerization and loses affinity for GM1-ganglioside. Here we report a new strategy, in which steric hindrance between CTB-antigen fusion subunits is significantly reduced through the integration of unfused CTB "molecular buffers" into the pentamer unit, making them more efficiently self-assemble into biologically active pentamers. In addition, the chimeric protein took a compact configuration, becoming small enough to be secreted, and one-step affinity-purified proteins, when administered through a mucosal route, induced specific immune responses in mice. Since our results are not dependent on the use of a particular expression system or vaccine antigen, this strategy could be broadly applicable to bacterial enterotoxin-based vaccine design.

Animals↗

PubMed1999

Secretion from bovine chromaffin cells acutely expressing exogenous proteins using a recombinant Semliki Forest virus containing an EGFP reporter.

Acute expression of recombinant proteins throughout a population of postmitotic bovine chromaffin cells was achieved using the Semliki Forest virus expression system (P. Liljestrom and H. Garoff (1991) Biotechnology 9:1356-1361). The virus was modified to express a green fluorescent protein, which faithfully reported the expression of the recombinant proteins. Two types of reporting virus were constructed: the first included a second subgenomic element, and the second an internal ribosome entry site. Both were used to express the recombinant proteins beta-galactosidase, 5HT3 receptor, or tetanus toxin light chain. Beta-galactosidase was used to quantify the rate of expression of recombinant protein in chromaffin cells, the 5HT3 receptor to trigger secretion, and the toxin to block secretion. The experiments clearly show that infection and expression of recombinant proteins throughout a population of chromaffin cells do not, per se, affect the rate and extent of triggered exocytosis, endocytosis, or membrane recycling pathways. The catecholamine content of the cell is unaltered, and the secretory mechanism can be accessed within a few hours after infection. This noncytopathic method of acutely expressing specific proteins at physiological levels in chromaffin cells offers a powerful new tool for dissecting the roles of many proteins implicated in exo- and endocytosis.

Animals↗

PubMed2005

Distribution and phenotype of murine rotavirus-specific B cells induced by intranasal immunization with 2/6 virus-like particles.

Virus-like particles containing the rotavirus (RV) internal proteins VP2 and VP6 (2/6-VLP) have been shown to induce serum and fecal antibodies as well as protection in mice after intranasal administration with a mutant of E. coli toxin, LT-R192G. To better understand the origin of fecal IgA induced by this protocol, we studied the RV-specific B cell response in systemic and mucosal lymphoid tissues using a flow cytometry assay that allows quantification and phenotypic characterization of RV-specific B lymphocytes. We also assessed the RV-specific antibody-secreting cells in the spleen and intestinal lamina propria (ILP). A remarkably high frequency of RV-specific B cells was found in the respiratory lymphoid tissues and spleen, of which only a minority expressed the alpha4beta7 integrin (intestinal homing receptor). In contrast, but in accordance with alpha4beta7 expression at the induction site, a very low response was observed in intestinal lymphoid tissues (mesenteric lymph nodes and ILP), which did not increase after a second immunization. Thus, intranasal immunization with a nonreplicating antigen does not induce an important number of RV-specific B cells with an intestinal homing profile.

Administration, Intranasal↗

PubMed1989

A comparison of bradykinin, angiotensin II and muscarinic stimulation of cultured bovine adrenal chromaffin cells.

Bradykinin, angiotensin II and a muscarinic agonist, acetyl-B-methacholine (methacholine) were all found to elicit catecholamine release from cultured bovine adrenal chromaffin cells. Bradykinin was the most potent of these secretagogues and methacholine the weakest, with angiotensin II intermediate in efficacy. All three secretagogues were much less effective than nicotinic stimulation. The three secretagogues all produced a rise in cytoplasmic free calcium concentration ([Ca2+]i), measured with the fluorescent indicator fura2, which was partially independent of external calcium. In the case of bradykinin the full rise in ([Ca2+]i) may involve a component of calcium entry in addition to release of calcium from an internal store. Secretion was also found to be partially independent of external calcium. The different efficacies of the three secretagogues in eliciting secretion were correlated with the rise in ([Ca2+]i) produced. The differing efficacies of the three secretagogues may be due to the extent of release of calcium from an intracellular store which itself is less effective in eliciting secretion than a rise in [Ca2+]i following calcium entry due to nicotine. Bradykinin also stimulates calcium entry, and this may increase the efficacy of the initial rise in [Ca2+]i. Treatment with pertussis toxin resulted in an enhancement of secretion in response to all of the secretagogues.

Adrenal Glands↗

PubMedDate not supplied

Botulinum C2 toxin ADP-ribosylates actin.

ADP-ribosylation of regulatory proteins is an important pathological mechanism by which various bacterial toxins affect eukaryotic cell functions. While diphtheria toxin catalyses the ADP-ribosylation of elongation factor 2, which results in inhibition of protein synthesis, cholera toxin and pertussis toxin ADP-ribosylate Ns and Ni, respectively, the GTP-binding regulatory components of the adenylate cyclase system, thereby modulating the bidirectional hormonal regulation of the adenylate cyclase. Botulinum C2 toxin is another toxin which has been reported to possess ADP-ribosyltransferase activity. This extremely toxic agent is produced by certain strains of Clostridium botulinum and induces hypotension, an increase in intestinal secretion, vascular permeability and haemorrhaging in the lungs. In contrast to botulinum neurotoxins, the botulinum C2 toxin apparently lacks any neurotoxic effects. Here we report that botulinum C2 toxin ADP-ribosylates a protein of relative molecular mass 43,000 (43K) in intact cells and in cell-free preparations. We present evidence that the 43K protein substrate is actin, which is apparently mono-ADP-ribosylated by the toxin. Botulinum C2 toxin also ADP-ribosylated purified liver G-actin, whereas liver F-actin was only poorly ADP-ribosylated and skeletal muscle actin was not ADP-ribosylated in either its G form or its F form. ADP-ribosylation of liver G-actin by botulinum C2 toxin resulted in a drastic reduction in viscosity of actin polymerized in vitro.

Actins↗

PubMed2005

Modulation of host cell signal transduction pathways by Helicobacter pylori infection.

Bacterial pathogens modulate host cell signal transduction responses to establish infection and cause disease. The purpose of the present summary, first presented at the Canadian Helicobacter Study Group meeting, is to discuss current knowledge of specific Helicobacter pylori factors, including the vacuolating cytotoxin, cytotoxin-associated gene A and the type four secretion system encoded by the cytotoxin-associated gene pathogenicity island and review the host cell signal transduction cascades that they modulate.

Antigens, Bacterial↗

PubMedDate not supplied

Role of the enteric nervous system in the pathophysiology of secretory diarrhea.

Details of the physiology and pathophysiology of epithelial secretion in the gastrointestinal tract are becoming clear, leading to new models of the mechanisms underlying diarrhea. The enteric nervous system is a critical component of the mechanism regulating fluid secretion in the normal gut and a key element in the pathophysiology of diarrhea. Neural reflex pathways increase epithelial fluid secretion in response to several enteric pathogens of veterinary importance such as Salmonella spp., Cryptosporidium parvum, rotavirus, and Clostridium difficile. Moreover, the enteric nervous system has an important role in epithelial secretion triggered by products of activated leukocytes during inflammation. New approaches targeting the enteric nervous system show promise for the treatment of secretory diarrhea.

Animal Diseases↗