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Results for “toxin secretion system”

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At least 253 records · Page 14Linked to original sources

PubMed1999

Role of actin depolymerization in the surfactant secretory response of alveolar epithelial type II cells.

Alveolar epithelial type II cells (AET2) respond with exocytosis of surfactant containing lamellar bodies to stimulation with mechanical stretch and secretagogues, a process that is fundamental for maintaining alveolar stability and lung gas exchange. In the present study in cultured rat AET2, we employed botulinum C2 toxin, a binary toxin which ADP ribosylates nonmuscle G-actin, as a specific tool to probe the role of the actin microfilament system in the surfactant secretory process. Incubation of AET2 with C2 toxin caused a dose-dependent decay of the cellular F-actin content to a minimum of 20% of baseline, concomitant with an increase in monomeric actin. In parallel, a significant augmentation of baseline surfactant secretion up to twofold elevated levels above control was noted, as assessed by the release of prelabeled phosphatidylcholine. Pretreatment with phalloidin, which stabilized F-actin and reduced the level of G-actin, prevented the C2 toxin-elicited enhancement of baseline surfactant secretion. Even low C2 toxin concentrations, resulting in a reduction of total cellular F-actin content of approximately 10%, sufficed to augment secretagogue (ATP) and, more impressively, mechanical stress elicited an increase in surfactant secretion; the response to the biophysical challenge more than doubled. When investigated in the absence of toxin, different secretagogues (ATP, phorbol ester, betamimetics) caused a rapid-onset, transient reduction of F-actin in the range between 15 and 25% as a consistent part of their secretory response pattern. These data suggest that the state of actin polymerization is intimately linked to the exocytosis process underlying surfactant secretion in AET2. Microfilament system-related compartmentalization effects and/or or the impact of the state of actin assembly on signaling events may be considered as underlying events.

Actins↗

PubMed2000

Sensitivity of embryonic rat dorsal root ganglia neurons to Clostridium botulinum neurotoxins.

Clostridium botulinum neurotoxins (BoNT) are zinc dependent endopeptidases which, once internalised into the neuronal cytosol, block neurotransmission by proteolysis of membrane-associated proteins putatively involved in synaptic vesicle docking and fusion with the plasma membrane. Although many studies have used a variety of cellular systems to study the neurotoxins, most require relatively large amounts of toxin or permeabilisation to internalise the neurotoxin. We present here a primary culture of embryonic rat dorsal root ganglia (DRG) neurons that exhibits calcium-dependent substance P secretion when depolarised with elevated extracellular potassium and is naturally BoNT sensitive. The DRG neurons showed a different IC50 for each of the toxins tested with a 1000 fold difference between the most and least potent neurotoxins (0.05, 0.3, 30 and approximately 60 nM for A, C, F and B, respectively). BoNT/A cleavage of SNAP-25 was seen as early as 2 h, but substance P secretion was not significantly inhibited until 4 h intoxication and the effects of BoNT/A were observed for as long as 15 days. This primary neuronal culture system represents a new and sensitive cellular model for the in vitro study of the botulinum neurotoxins.

Animals↗

PubMed1989

Renal epithelial cyst formation and enlargement in vitro: dependence on cAMP.

Cysts, a common abnormality of kidneys, are collections of urine-like fluid enclosed by a continuous layer of epithelial cells. Renal cysts derive from nephrons and collecting ducts and progressively enlarge as a consequence of epithelial proliferation and transepithelial fluid secretion. The initiation of cyst formation and the factors that control cyst enlargement are unknown. We used an in vitro model of renal cysts to explore the role of the cAMP signal transduction system in the formation and expansion of cysts. MDCK cells, cultured in hydrated-collagen gel, produced polarized monolayered epithelial cysts when intracellular cAMP was increased by prostaglandin E1, arginine vasopressin, cholera toxin, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate. All agonists were potentiated by 3-isobutyl-1-methylxanthine, a nucleotide phosphodiesterase inhibitor. The cell proliferation component of cyst enlargement was accelerated by cAMP agonists, as shown by the increased growth of MDCK cells in subconfluent monolayers. The fluid secretion component, reflected by the transepithelial movement of fluid across polarized monolayers of MDCK cells grown on permeable supports, was stimulated by cAMP agonists in the basolateral medium. Chloride levels were higher in the cyst fluid and the secreted fluid than in the bathing medium. We conclude that the development of MDCK cysts is dependent on cAMP. This signal transduction system may be an important modulator of epithelial cell proliferation and transepithelial fluid secretion in the kidney.

1-Methyl-3-isobutylxanthine↗

PubMed1988

Oral administration of cholera toxin-Sendai virus conjugate potentiates gut and respiratory immunity against Sendai virus.

Successful oral immunization to prevent infectious diseases in the gastrointestinal tract as well as distant mucosal tissues may depend on the effectiveness of an Ag to induce gut immune responses. We and others have previously reported that cholera toxin possesses strong adjuvant effects on the gut immune response to co-administered Ag. To explore further adjuvant effects of cholera toxin, the holotoxin or its B subunit was chemically cross-linked to Sendai virus. The resulting conjugates, which were not infectious, were evaluated for their capacity to induce gut immune responses against Sendai virus after oral administration to mice. Conjugating cholera toxin to virus significantly enhanced the adjuvant activity of cholera toxin compared to simple mixing. Cholera toxin B subunit, however, did not show an adjuvant effect either by itself or conjugated with the virus. Oral administration of the Sendai virus-cholera toxin conjugate was also able to prime for protective anti-viral responses in the respiratory tract. Mice that were orally immunized with the conjugate and intra-nasally boosted with inactivated virus alone showed virus-specific IgA titers in nasal secretions that correlated with protection against direct nasal challenge with live Sendai virus. For comparison, s.c. immunization was also studied. Systemic immunization with the virus-cholera toxin conjugate induced virus-specific antibody responses in serum as well as in the respiratory tract but failed to protect the upper respiratory tract against virus challenge. Systemic immunization plus an intra-nasal boost did, however, confer a variable degree of protection to the upper respiratory tract, which correlated primarily with bronchoalveolar lavage (lung) antibody titers.

Adjuvants, Immunologic↗

PubMed2004

Lysophospholipase A activity of Pseudomonas aeruginosa type III secretory toxin ExoU.

Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU that is delivered directly into the eukaryotic cell via the type III secretion system. Recent studies demonstrated that ExoU has lipase activity, and that the cytotoxicity of ExoU is dependent on its patatin-like phospholipase domain. We investigated the phospholipase A (PLA) activity of ExoU. ExoU, but not non-catalytic ExoU-S142A, preincubated with the BEAS-2B cell lysate showed a weak increase of Ca(2+)-independent PLA(2) activity. When activated ExoU was mixed with secretory type PLA(2), more phospholipase activity was observed, suggesting that ExoU has lysophospholipase A (lysoPLA) activity. A significant increase in lysoPLA activity was also observed. Glycerol enhanced this activity and inhibitors of iPLA(2) suppressed ExoU's lysoPLA activity. Our results suggest that ExoU has a potent lysoPLA activity that requires the presence of the catalytically active site Ser(142) with an unknown eukaryotic cell factor(s) for its activation.

Adenosine Triphosphate↗

PubMed1994

[Effect of phorbol ester on tissue-type plasminogen activator (t-PA) secretion in endometrial carcinoma cell line in vitro].

A plasminogen activator (PA) system is involved in ovulation, implantation, tumor invasion and metastasis. In order to clarify the regulation of this PA system in endometrial cells, we examined which agent affecting cellular function altered tissue-type plasminogen activator (t-PA) secretion by endometrial carcinoma cell line (KLE cells) in vitro. Triiodothyronine, retinoic acid, insulin, 8-bromo-cAMP, PDGF, IGF-I, basic FGF or TNF-alpha did not alter t-PA secretion while the activator of protein kinase C, phorbol myristate acetate (PMA) stimulated t-PA secretion in a dose-dependent fashion (10(-10)-10(-8) M). The time required to give a statistically significant increase in t-PA over control was 3 hours, and the maximal increase was seen after 24 hours of exposure. Another active phorbol ester, PDD also stimulated t-PA secretion while inactive forms of phorbol ester, 4 alpha-PDD and phorbol did not alter it. Cholera toxin or 8-bromo-cAMP did not affect t-PA secretion, but enhanced PMA-stimulated t-PA secretion. Cycloheximide and actinomycin D completely abolished PMA-stimulated t-PA secretion. These results suggest that (1) t-PA secretion in the endometrial carcinoma cell is modulated by a protein kinase C system, (2) This effect is through new RNA production and protein synthesis. (3) There is a complicated relationship between protein the kinase C and protein kinase A system as to the regulation of t-PA secretion. This would be a suitable model to clarify the PA system in endometrial cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1998

Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems.

A decade ago, Pugsley and colleagues reported the existence of a large region of Klebsiella DNA, distinct from the Klebsiella gene encoding pullulanase, which was necessary for secretion of this enzyme to the cell surface in Escherichia coli (d'Enfert et al., 1987a,b). The pul genes it contained proved to be the tip of an iceberg. The sequences reported before 1992 (d'Enfert et al., 1987a,b; d'Enfert & Pugsley, 1989; Pugsley & Reyss, 1990; Reyss & Pugsley, 1990) included only one gene (pulD) that matched any sequence in the data base; a 220 amino acid residue segment of PulD was 32% identical with a portion of the filamentous phage-encoded protein, pIV. But by the time the sequence of the 18.8 kb DNA fragment that contained the pul genes had been completed (Possot et al., 1992), reports of sets of homologous genes in several species of Gram-negative plant and animal pathogens had appeared. For the most part, these gene clusters were cloned by their ability to complement mutants that produced, but failed to secrete, proteins normally found in the extracellular milieu; when tested, the mutants showed reduced pathogenicity or were totally avirulent. The secreted proteins included hydrolytic enzymes such as cellulase and pectinase from plant pathogens, and proteases and toxins from animal pathogens. The multi-gene family necessary for secretion of these enzymes is now known as the type II system or the main terminal branch (MTB) of the general secretion pathway (GSP). As summarized by Pugsley et al. (1997), the current tally includes type II systems from Klebsiella oxytoca (pul), Erwinia chrysanthemi and carotovora (out), Xanthomonas campestris (xps), Pseudomonas aeruginosa (xcp), Aeromonas hydrophila (exe), and Vibrio cholerae (eps). A second type II system (sps) necessary for deposition of the S-layer on the cell surface in A. hydrophila is more similar to the X. campestris than A. hydrophila genes (Thomas & Trust, 1995). The biggest surprise has been the discovery of a complete set of type II secretion genes in E. coli K12. The E. coli genes are not expressed under normal growth conditions, and a search is underway to find inducing conditions and secretion substrates (Francetic & Pugsley, 1996). Impressive progress has already been made in defining components of the pathway. What remains to be understood in mechanistic detail is how this protein secretion system functions.

Bacterial Proteins↗

PubMed1994

Exploration of mucosal immunity in humans: relevance to vaccine development.

Although the immune system is remarkably diverse, there is evidence that certain types of immune responses take place and are restricted to certain anatomic locations within the body. The concept of a common mucosal immune system that provides immune reactivity not only at the site of antigen deposition but also at remote mucosal sites may be explained by the utilization of organ-specific recognition molecules by circulating precursors of mucosal immunoblasts and by the production of certain maturation factors (e.g. cytokines, hormones) produced preferentially in certain organs or parts of a given organ. This notion may explain the unification of immune responses in diverse mucosal sites and the physiologic segregation of mucosal from systemic immune mechanisms. Novel methods have been developed to enable studies of antigen specific B and T cell responses in various mucosal and extramucosal tissues in primates and rodents, using cholera toxin or its B subunit as prototype immunogens and mucosal carrier-delivery systems. The tissue localization and isotype commitment of antibody-secreting cells (ASC) and the homing potential of their circulating precursors have also been examined after oral, nasal, intra-tonsillar, rectal and/or genital immunization(s). The anatomical distribution of T- and accessory cell-derived cytokines has also been examined. These tools and approaches are being employed in studies attempting to induce optimal mucosal immune responses to several mucosal pathogens including HIV-1, in certain organs such as the lower gastrointestinal tract and the female urogenital tract.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Genetic and molecular approaches to synthesis and action of the yeast killer toxin.

The K1 killer toxin of Saccharomyces cerevisiae is a secreted, virally-coded protein lethal to sensitive yeasts. Killer yeasts are immune to the toxin they produce. This killer system has been extensively examined from genetic and molecular perspectives. Here we review the biology of killer yeasts, and examine the synthesis and action of the protein toxin and the immunity component. We summarise the structure of the toxin precursor gene and its protein products, outline the proteolytic processing of the toxin subunits from the precursor, and their passage through the yeast secretory pathway. We then discuss the mode of action of the toxin, its lectin-like interaction with a cell wall glucan, and its probable role in forming channels in the yeast plasma membrane. In addition we describe models of how a toxin precursor species functions as the immunity component, probably by interfering with channel formation. We conclude with a review of the functional domains of the toxin structural gene as determined by site-directed mutagenesis. This work has identified regions associated with glucan binding, toxin activity, and immunity.

Cell Membrane↗

PubMed2003

Regulation, secretion and activity of type III-secreted proteins of enterohaemorrhagic Escherichia coli O157.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 causes gastrointestinal disease with the potential for life-threatening sequelae. Although Shiga-like toxins are responsible for much of the serious pathology in humans, the bacterium also possesses a type III protein secretion system that is responsible for intimate attachment to host intestinal mucosa. This sophisticated interaction requires co-ordination that is governed by environmental and genetic factors. Ongoing research supports the following model for how EHEC enables and controls this process: (i) specific environmental cues that are present in the host result in the expression of a number of adhesins, including fimbriae, which allow the initial binding to the mucosal surface. The same conditions support the expression of the basal type III secretion apparatus; (ii) targeting and assembly of the translocon requires both an mRNA signal and chaperones, with coupled translation and secretion of translocon proteins, EspA, B and D; (iii) opening up of a conduit between the bacterium and host cell releases a cytoplasmic pool of effector proteins. A consequence of this is increased expression of particular effector proteins. Potentially, different proteins could be released into the cell at different times or have activities modulated with time; (iv) intimate contact between the translocated intimin receptor (Tir) and the bacterial surface factor intimin requires translocon expression to be down-regulated and translocon filaments to be lost. Fluorescent protein fusions allow contact-mediated regulation and protein targeting through the type III secretion system to be studied in detail.

Adhesins, Bacterial↗

PubMed2002

Staphylococcal alpha-toxin synergistically enhances inflammation caused by bacterial components.

This study was performed to investigate the in vivo effects of staphylococcal alpha-toxin on phagocytosis and the secretion of proinflammatory cytokines at local sites of intraperitoneal toxin-challenged mice. A dosage of 45 hemolytic units (HU) of alpha-toxin induced a marked increase in the peritoneal neutrophil count. The toxin caused a 52% decrease in phagocytosis by peritoneal macrophages, compared with that of control mice receiving Staphylococcus aureus particles alone. However, no effect on phagocytosis in neutrophils was observed. A dosage of 45 HU toxin and the synergistic activity of S. aureus particles strongly induced interleukin (IL) 6 secretion but only mildly induced IL-1alpha secretion. The toxin did not induce the secretion of tumor necrosis factor-alpha (TNF-alpha). Interestingly, S. aureus culture supernatant induced the secretion of TNF-alpha in cultured macrophages. These results suggest that alpha-toxin damages the primary host defense system by inducing the oversecretion of IL-1alpha and IL-6, but not TNF-alpha, via a mechanism that requires the synergistic action of bacterial components.

Animals↗

PubMed2005

Effect of alcohol consumption on the gut.

Both acute and chronic alcohol consumption have severe effects on the structure and function of the entire gastrointestinal tract (GIT) which result in a vicious cycle. The healthy person who begins to drink heavily, first experiences the toxic effects of high concentrations of ethanol. Mucosal damage compromises the basic functions of the GIT. Suppression of the gastrointestinal immune system and increased transport of toxins across the mucosa result in increased susceptibility to infections. Inhibition of digestion, absorption and secretion cause diarrhea and reduce the transfer of nutrients to the rest of the body. As the individual becomes more dependent on alcohol, the functional reserve and regenerative capacity of the GIT are overwhelmed and malnutrition increases. The rate of progression of this cycle depends on several factors including nutritional intake. Whilst the clinical effects of alcohol are well recognized, more research is required to fully elucidate the underlying mechanisms.

Alcohol Drinking↗

PubMed1998

Ranakinin, a naturally occurring tachykinin, stimulates phospholipase C activity in the frog adrenal gland.

We have previously shown that the frog adrenal gland is innervated by a dense network of fibers containing ranakinin, one of the endogenous tachykinins in the amphibian Rana ridibunda, and we have found that ranakinin stimulates in vitro corticosteroid secretion by frog adrenal tissue. To elucidate the mechanism of action of ranakinin on the frog adrenal gland, we investigated the effect of ranakinin on cAMP formation and polyphosphoinositide metabolism. Incubation of frog adrenal explants with various tachykinins, including ranakinin, substance P, neurokinin A, or neurokinin B, did not produce any significant modification of cAMP concentrations. In contrast, ranakinin induced a time- and dose-dependent stimulation of inositol phosphate formation with a concomitant decrease in membrane polyphosphoinositides. Pretreatment of the tissue slices with the phospholipase C inhibitor U-73122 or with pertussis toxin completely abolished the stimulatory effect of ranakinin on inositol phosphate formation. Prolonged administration of U-73122 to perifused frog adrenal explants markedly attenuated the ranakinin-evoked stimulation of corticosterone and aldosterone secretion. Taken together, these data indicate that in the frog adrenal gland, ranakinin has no effect on the adenylyl cyclase system, but enhances polyphosphoinositide hydrolysis. The stimulatory action of ranakinin on inositol phosphate formation and corticosteroid secretion is mediated through activation of a phospholipase C positively coupled to a pertussis toxin-sensitive G protein.

Adrenal Cortex Hormones↗

PubMed2006

A Helicobacter pylori vacuolating toxin mutant that fails to oligomerize has a dominant negative phenotype.

Most Helicobacter pylori strains secrete a toxin (VacA) that causes massive vacuolization of target cells and which is a major virulence factor of H. pylori. The VacA amino-terminal region is required for the induction of vacuolization. The aim of the present study was a deeper understanding of the critical role of the N-terminal regions that are protected from proteolysis when VacA interacts with artificial membranes. Using a counterselection system, we constructed an H. pylori strain, SPM 326-Delta49-57, that produces a mutant toxin with a deletion of eight amino acids in one of these protected regions. VacA Delta49-57 was correctly secreted by H. pylori but failed to oligomerize and did not have any detectable vacuolating cytotoxic activity. However, the mutant toxin was internalized normally and stained the perinuclear region of HeLa cells. Moreover, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. This loss of activity was correlated with the disappearance of oligomers in electron microscopy. These findings indicate that the deletion in VacA Delta49-57 disrupts the intermolecular interactions required for the oligomerization of the toxin.

Bacterial Proteins↗

PubMed1995

Intestinal immune response induced in mice by staphylococcal enterotoxin B.

To investigate the induction of intestinal immunity to staphylococcal enterotoxins (SE) we have chosen the mouse as an experimental model. Since this species is devoid of emetic mechanism, SE can be administered orally without any loss. Mice were treated orally and/or parenterally with staphylococcal enterotoxin B (SEB). The anti-SEB response, either in serum or in the supernatant of in vitro cultured intestinal fragments was determined by enzyme immunoassay (EIA). The results showed that orally given SEB induced specific antibodies both in serum and intestinal secretions. Compared to oral route alone, parenteral followed by oral administration of SEB induced a higher intestinal response with IgA as predominant isotype. Although these results cannot directly be extrapolated to humans or animals with emetic reaction to SE, they do show the implication of intestinal immune system in response to this group of toxins.

Animals↗

PubMed2000

Convergence of the secretory pathways for cholera toxin and the filamentous phage, CTXphi.

Virulence of Vibrio cholerae depends on secretion of cholera toxin (CT), which is encoded within the genome of a filamentous phage, CTXphi. Release of CT is mediated by the extracellular protein secretion (eps) type II secretion system. Here, the outer membrane component of this system, EpsD, was shown to be required for secretion of the phage as well. Thus, EpsD plays a role both in pathogenicity and in horizontal transfer of a key virulence gene. Genomic analysis suggests that additional filamentous phages also exploit chromosome-encoded outer membrane channels.

Bacterial Outer Membrane Proteins↗

PubMed1985

The enteric nervous system participates in the secretory response to the heat stable enterotoxins of Escherichia coli in rats and cats.

Intestinal secretion was evoked in periarterially denervated jejunal segments of anesthetized rats and cats by exposing the intestines to the heat stable (ST) toxins from a strain of Escherichia coli producing both STa and STb toxins. The secretion was significantly inhibited and to about the same relative extent by the addition of each one of the three following drugs: hexamethonium (i.v., rats), lidocaine (applied on the serosal surface, rats) and tetrodotoxin (intra-arterial, cats). Atropine inhibited fluid secretion in some experiments. It is proposed that a nervous mechanism is mediating part of the secretory response to Escherichia coli heat stable toxins, since three different drugs, which influence nervous activity in different ways, significantly diminished the secretory response. A model for the secretory nervous reflex(es) within the enteric nervous system is proposed; Escherichia coli heat stable toxins activate a "receptor cell" in the epithelium, which then stimulates surrounding dendritic nerve endings via the release of unknown substance(s). A nicotinic receptor is involved but further characteristics of the nervous reflex(es) remain to be elucidated.

Animals↗

PubMed1978

Effect of heat stable and heat labile Escherichia coli enterotoxins and cholera toxin in combination with theophylline on unidirectional sodium and chloride flux in the small intestine of weanling swine.

The effect of heat stable and heat labile Escherichia coli enterotoxins or cholera toxin in combination with theophylline on net water, sodium and chloride and unidirectional sodium and chloride fluxes was examined in acute isolated loops of jejunum of weanling swine. The effect of heat stable enterotoxin in combination with theophylline was determined in loops located in the proximal jejunum, while combinations of theophylline and either heat labile enterotoxin or cholera toxin were studied in the distal jejunum. In each situation the addition of theophylline resulted in an additive rather than a synergistic increment of intestinal secretory activity. This study implies that the intestinal adenyl cyclase system and enterotoxin induced intestinal secretion may not be directly related in the swine small intestine.

Animals↗