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Results for “toxin secretion system”

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PubMed1989

Evidence for an intramural nervous control of epithelial cell migration in the small intestine of the rat.

The migration of epithelial cells along the crypt-villus axis in the small intestine of the rat was followed by labelling epithelial cells during mitosis with [3H]thymidine given i.v. Using two different techniques (autoradiography and determination of tissue radioactivity) it was demonstrated that 6-9 h after giving the tracer the labelled cells had migrated longer in intestinal segments exposed to cholera toxin than in control segments. This effect of cholera toxin was abolished by giving hexamethonium. We have earlier shown that cholera toxin induces fluid secretion to a large extent by activating the enteric nervous system and we conclude from the present observations that cholera toxin in a similar fashion exerts a trophic effect on the intestinal epithelium via intramural nervous reflexes. The importance of co-release of several neurotransmitters in explaining the trophic effect is tentatively discussed.

Animals↗

PubMed2005

Exploring cargo transport mechanics in the type IV secretion systems.

Type IV secretion systems (T4SSs) are used by various bacteria to deliver protein and DNA molecules to a wide range of target cells. These include systems that are directly involved in pathogenesis, such as the secretion of pertussis toxin by Bordetella pertussis into human cells and the delivery of single-stranded DNA (ssDNA) into plants by Agrobacterium. These complex systems are composed of proteins that span the bacterial cytoplasm. The Agrobacterium T4SS is composed of 12 virulence proteins and delivers its transferred ssDNA and several virulence protein substrates to a variety of eukaryotic cells. Recent studies on the Agrobacterium T4SS have revealed new information on the localization and structure of its proteins in the bacteria, the biochemical properties of its transport signal, the route of a DNA substrate through the secretion system, and the initial point of contact of the system with its host. These findings have expanded our knowledge and understanding of the still mostly obscure structure and function of the T4SSs.

Bacteria↗

PubMed1995

The pathogenesis of experimental toxic shock syndrome: the role of interleukin-2 in the induction of hypotension and release of cytokines.

Toxic shock syndrome (TSS) is a multisystem disorder characterized by fever, hypotension, and involvement of three other organ systems. The etiologic agent is a toxigenic strain of Staphylococcus aureus which secretes the exotoxin, TSST-1. The toxin is a superantigen which stimulates the immune system to produce interleukin-1 (IL-1), interleukin-2, and tumor necrosis factor (TNF). We hypothesized that TSST-1 induces the release of IL-2 which in turn is either directly involved or acts via an additional mediator to produce hypotension. We submitted four pairs of normal anesthetized adult female baboons to intravenous boluses of TSST-1. One baboon in each pair received anti-IL-2 intravenously and anti-IL-2 receptor intrathyroidally 15 min prior to TSST-1. The other baboon received the same dose and placement of anti-sheep red blood cell antibody. Systolic and diastolic blood pressure was recorded continuously and mean arterial pressure was calculated and plotted. IL-1, IL-2, IL-6, and TNF were measured in serum at varying times before and after toxin administration. Systolic, diastolic, and mean arterial pressure were significantly lower in the sham-treated group versus the experimental (anti-IL-2/IL-2R) group (p < .05 for all variables). In addition no differences were seen in any of the measurements between experimentally treated baboons and those receiving no TSST-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2005

Attenuated virulence of an Aeromonas salmonicida subsp. salmonicida type III secretion mutant in a rainbow trout model.

Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a severe systemic disease affecting salmonid fish. This bacterium contains a type III protein secretion system that is responsible for the secretion and translocation of the ADP-ribosylating toxin, AexT, into the cytosol of fish cells. This study showed that inactivation of the type III secretion system by marker-replacement mutagenesis of the gene ascV, which encodes an inner-membrane component of the type III secretion system, attenuated virulence in a rainbow trout model. The isogenic ascV deletion mutant was phagocytosed by peripheral blood leukocytes but the wild-type (wt) A. salmonicida subsp. salmonicida isolate was not. Histological examination of fish experimentally infected with the wt bacterium revealed extensive tissue necrosis and bacterial aggregates in all organs examined, including the heart, kidney and liver, indicating that the isolate established a systemic infection. Cumulative mortality of fish experimentally infected with the wt bacterium reached 88%. In contrast, no mortality was observed among fish infected with the same dose of the ascV mutant, and histological examination of fish infected with this strain revealed healthy organs. The results indicate that the type III secretion system of A. salmonicida subsp. salmonicida is required to establish systemic infection.

Aeromonas salmonicida↗

PubMed2006

Salmonella enterica SpvB ADP-ribosylates actin at position arginine-177-characterization of the catalytic domain within the SpvB protein and a comparison to binary clostridial actin-ADP-ribosylating toxins.

The SpvB protein from Salmonella enterica was recently discovered as an actin-ADP-ribosylating toxin. SpvB is most likely delivered via a type-III secretion system into eukaryotic cells and does not have a binding/translocation component. This is in contrast to the family of binary actin-ADP-ribosylating toxins from various Bacillus and Clostridium species. However, there are homologies in amino acid sequences between the C-terminal domain of SpvB and the catalytic domains of the actin-ADP-ribosylating toxins such as C2 toxin from Clostridium botulinum and iota toxin from Clostridium perfringens. We compared the biochemical properties of the catalytic C-terminal domain of SpvB (C/SpvB) with the enzyme components of C2 toxin and iota toxin. The specificity of C/SpvB concerning the modification of G- or F-actin was comparable to the C2 and iota toxins, although there were distinct differences regarding the recognition of actin isoforms. C/SpvB and iota toxin modify both muscle alpha-actin and nonmuscle beta/gamma-actin, whereas C2 toxin only modifies beta/gamma-actin. In contrast to the iota and C2 toxins, C/SpvB possessed no detectable glycohydrolase activity in the absence of a protein substrate. The maximal reaction rates were comparable for all toxins, whereas variable K(m) values for NAD were evident. We identified arginine-177 as the modification site for C/SpvB with the actin homologue protein Act88F from Drosophila.

ADP Ribose Transferases↗

PubMed2005

Site-specific antiphagocytic function of the Photorhabdus luminescens type III secretion system during insect colonization.

Photorhabdus is an entomopathogenic bacterium belonging to the Enterobacteriaceae. The genome of the TT01 strain of Photorhabdus luminescens was recently sequenced and a large number of toxin-encoding genes were found. Genomic analysis predicted the presence on the chromosome of genes encoding a type three secretion system (TTSS), the main role of which is the delivery of effector proteins directly into eukaryotic host cells. We report here the functional characterization of the TTSS. The locus identified encodes the secretion/translocation apparatus, gene expression regulators and an effector protein - LopT - homologous to the Yersinia cysteine protease cytotoxin YopT. Heterologous expression in Yersinia demonstrated that LopT was translocated into mammal cells in an active form, as shown by the appearance of a form of the RhoA GTPase with modified electrophoretic mobility. In vitro study showed that recombinant LopT was able to release RhoA and Rac from human and insect cell membrane. In vivo assays of infection of the cutworm Spodoptera littoralis and the locust Locusta migratoria with a TT01 strain carrying a translational fusion of the lopT gene with the gfp reporter gene revealed that the lopT gene was switched on only at sites of cellular defence reactions, such as nodulation, in insects. TTSS-mutant did not induce nodule formation and underwent phagocytosis by insect macrophage cells, suggesting that the LopT effector plays an essential role in preventing phagocytosis and indicating an unexpected link between TTSS expression and the nodule reaction in insects.

Amino Acid Sequence↗

PubMed2004

Systematic analysis, by the yeast two-hybrid, of protein interaction between components of the type II secretory machinery of Erwinia chrysanthemi.

Type II systems allow for the secretion of numerous enzymes and toxins in several Gram-negative pathogens. In Erwinia chrysanthemi, 14 Out proteins are necessary for building the type II apparatus. We performed a systematic two-hybrid analysis to test interactions between the periplasmic regions of the Out proteins. Results obtained using this approach suggested that OutJ (a pseudopilin) was able to interact with (i) OutD, the outer membrane secretin, (ii) OutI, mainly located in the periplasm, and (iii) OutL, an inner membrane protein. Taken together, these results suggest that OutJ is involved in multiple partnerships. Implications of these partnerships in the overall architecture of the type II secretion machinery are discussed.

Bacterial Outer Membrane Proteins↗

PubMed2006

Growth phase regulation of Vibrio cholerae RTX toxin export.

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, secretes several "accessory" toxins, including RTX toxin, which causes the cross-linking of the actin cytoskeleton. RTX toxin is exported to the extracellular milieu by an atypical type I secretion system (T1SS), and we previously noted that RTX-associated activity is detectable only in supernatant fluids from log phase cultures. Here, we investigate the mechanisms for regulating RTX toxin activity in supernatant fluids. We find that exported proteases are capable of destroying RTX activity and may therefore play a role in the growth phase regulation of toxin activity. We determined that the absence of RTX toxin in stationary-phase culture supernatant fluids is also due to a lack of toxin secretion and not attributable to solely proteolytic degradation. We ascertained that the T1SS apparatus is regulated at the transcriptional level by growth phase control that is independent of quorum sensing, unlike other virulence factors of V. cholerae. Additionally, in stationary-phase cultures, all RTX toxin activity is associated with bacterial membranes or outer membrane vesicles.

Bacterial Toxins↗

PubMed1987

Effect of systemic acid-base balance on ileal secretion.

We previously reported that systemic pH and HCO3 concentration affect ileal water and electrolyte absorption. To determine whether these effects could influence an ongoing secretory process, we measured transport in ileal loops exposed to either saline or 50-75 micrograms cholera toxin in mechanically ventilated Sprague-Dawley rats anesthetized with pentobarbital sodium. The effects of acute respiratory and metabolic acidosis and alkalosis were then examined. Decreases in systemic pH during respiratory acidosis caused equivalent increases in net water (54 +/- 8 microliters . cm-1 . h-1) and Na absorption (7 +/- 1 mu eq . cm- . h-1) and smaller increases in Cl absorption in cholera toxin compared with saline loops. These increases reversed the net secretion of these ions observed during alkalemia in the cholera toxin loops to net absorption. Metabolic acidosis and alkalosis and respiratory compensation of systemic pH of these metabolic disorders also altered cholera toxin-induced secretion in a direction consistent with the pH change. The increase in net HCO3 secretion caused by cholera toxin was unaffected by the respiratory disorders and did not vary with the HCO3 concentration in the metabolic disorders. These findings suggest that the systemic acid-base disorders that characterize intestinal secretory states may themselves alter intestinal absorptive function and fluid losses.

Acid-Base Equilibrium↗

PubMed2002

Membrane localization of the S1 subunit of pertussis toxin in Bordetella pertussis and implications for pertussis toxin secretion.

Pertussis toxin is secreted from Bordetella pertussis with the assistance of the Ptl transport system, a member of the type IV family of macromolecular transporters. The S1 subunit and the B oligomer combine to form the holotoxin prior to export from the bacterial cell, although the site of assembly is not known. To better understand the pathway of pertussis toxin assembly and secretion, we examined the subcellular location of the S1 subunit, expressed with or without the B oligomer and the Ptl proteins. In wild-type B. pertussis, the majority of the S1 subunit that remained cell associated localized to the bacterial membranes. In mutants of B. pertussis that do not express pertussis toxin and/or the Ptl proteins, full-length S1, expressed from a plasmid, partitioned almost entirely to the bacterial membranes. Several lines of evidence strongly suggest that the S1 subunit localizes to the outer membrane of B. pertussis. First, we found that membrane-bound full-length S1 was almost completely insoluble in Triton X-100. Second, recombinant S1 previously has been shown to localize to the outer membrane of Escherichia coli (J. T. Barbieri, M. Pizza, G. Cortina, and R. Rappuoli, Infect. Immun. 58:999-1003, 1990). Third, the S1 subunit possesses a distinctive amino acid motif at its carboxy terminus, including a terminal phenylalanine, which is highly conserved among bacterial outer membrane proteins. By using site-directed mutagenesis, we determined that the terminal phenylalanine is critical for stable expression of the S1 subunit. Our findings provide evidence that prior to assembly with the B oligomer and independent of the Ptl proteins, the S1 subunit localizes to the outer membrane of B. pertussis. Thus, outer membrane-bound S1 may serve as a nucleation site for assembly with the B oligomer and for interactions with the Ptl transport system.

Biological Transport↗

PubMed1987

Morphology and secretory activity of digitonin- and alpha-toxin-permeabilized chromaffin cells.

An ultrastructural examination of cultured bovine chromaffin cells permeabilized with Staphylococcus aureus alpha-toxin or digitonin revealed differences in the preservation of cell morphology. The toxin-treated cells closely resembled control cultured cells whereas digitonin-treated cells showed gradations in cytoplasmic densities suggesting extraction, some swelling of the endoplasmic reticulum and, occasionally, discontinuities in the plasma membrane and free granules in the extracellular medium. In both cell models, there was a swelling of the mitochondria. Horseradish peroxidase labelling of permeabilized cells marked the cytoplasm of digitonin-treated cells but only the surface of toxin-treated cells, demonstrating that larger lesions were caused by digitonin. In stimulated cells, the decrease in volumetric density of chromaffin granules correlated well with catecholamine release. The sites of secretory activity could be demonstrated in toxin-treated cells using horseradish peroxidase as a surface marker. Although both cell systems secrete catecholamines in response to calcium stimulation, their calcium requirements and the kinetics of release were different. In alpha-toxin-treated cells, 100 microM free calcium induced maximal catecholamine release. In digitonin-treated cells, 20 microM evoked maximal release but secretion was blocked at 100 microM. Catecholamine release terminated in digitonin-treated cells within 10 min but continued in alpha-toxin-treated cells for at least 60 min. In addition, the maximal release observed in toxin-treated cells (50%) was always greater than that observed in digitonin-permeabilized cells (20%). The results suggest that both exocytosis and granule translocation are operational in alpha-toxin-treated cells, but that the translocation step or the docking of granules at the plasma membrane may be impaired in digitonin-treated cells.

Adrenal Medulla↗

PubMed2004

BTLCP proteins: a novel family of bacterial transglutaminase-like cysteine proteinases.

Using sequence similarity searches and top-of-the-range fold-recognition methods, we have identified a novel family of bacterial transglutaminase-like cysteine proteinases (BTLCPs) with an invariant Cys-His-Asp catalytic triad and a predicted N-terminal signal sequence. This family of previously uncharacterized hypothetical proteins encompasses sequences of unknown function from DUF920 (in the Pfam database) and COG3672. BTLCPs are predicted to possess the papain-like cysteine proteinase fold and catalyze post-translational protein modification through transamidase, acetylase or hydrolase activity. Inspection of neighboring genes encoding BTLCPs suggests a link between this predicted activity and a type-I secretion system resembling ATP-binding cassette exporters of toxins and proteases involved in bacterial pathogenicity.

Amino Acid Sequence↗

PubMed2002

Development of a method for heterologous gene expression in Enterobacter amnigenus, a potential host for the biological control of mosquito larvi.

An integrative plasmid containing a 1.3 kb fragment of chromosomal DNA from Enterobacter amnigenus was constructed. The Omega fragment encoding spectinomycin/streptomycin resistance was cloned into the unique BglII site of the resulting plasmid, and the interrupted fragment was transferred via plasmid pMAK705 by electroporation into E. amnigenus with a selection for spectinomycin resistance. Cointegrants were resolved to generate an E. amnigenus strain that expressed spectinomycin resistance, but grew as rapidly as the parental strain. The cloned fragment encodes a putative homologue of the proW gene of Escherichia coli that is not essential for E. amnigenus growth. The integrative plasmid is now available to introduce any heterologous DNA into the E. amnigenus chromosome, for the construction of promoter-probe vectors for the studies of gene regulation, or to construct plasmids suitable for the isolation of secretion signals. Immediate applications of this system will include the expression and secretion of crystal toxins from bacilli for the biological control of mosquito larvae infected with the bacterial host.

ATP-Binding Cassette Transporters↗

PubMed2002

2001: a year of major advances in anthrax toxin research.

Anthrax is caused when spores of Bacillus anthracis enter a host and germinate. The bacteria multiply and secrete a tripartite toxin causing local edema and, in systemic infection, death. In nature, anthrax is primarily observed in cattle and other herbivores; humans are susceptible but rarely affected. In 2001, anthrax spores were used effectively for the first time in bioterrorist attacks, resulting in 11 confirmed cases of human disease and five deaths. These events have underscored the need for improved prophylaxis, therapeutics and a molecular understanding of the toxin. The good news about anthrax is that several decisive discoveries regarding the toxin have been reported recently. Most notably, the toxin receptor was identified, the 3-D structures of two of the toxin subunits were solved and potent in vivo inhibitors were designed. These findings have improved our understanding of the intoxication mechanism and are stimulating the design of strategies to fight disease in the future.

Animals↗

PubMed2003

Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia.

Overall, hospital-acquired pneumonia (HAP) caused by Pseudomonas aeruginosa is associated with high attributable mortality. Although the intrinsic virulence of P. aeruginosa undoubtedly contributes to this phenomenon, it is unclear whether all strains share this property or whether only a subpopulation of strains are capable of causing such severe disease. In this study, the virulence of 35 P. aeruginosa isolates obtained from patients with HAP by use of a cytolytic cell-death assay, an apoptosis assay, and a mouse model of pneumonia. The virulence of individual isolates differed significantly from one to another in each of these assays. Increased virulence was associated with the secretion of ExoU, a toxin transported by the P. aeruginosa type III secretion system. Secretion of ExoS or ExoY, 2 other proteins transported by this system, was not consistently associated with increased virulence. Together, these findings suggest that secretion of ExoU is a marker for highly virulent strains of P. aeruginosa.

Animals↗

PubMed2006

Effects of the type III secreted pseudomonal toxin ExoS in the yeast Saccharomyces cerevisiae.

Pseudomonas aeruginosa secretes a number of toxins by a type III system, and these are important in virulence. One of them, ExoS, is a bifunctional toxin, with a GTPase-activating protein domain, as well as ADP ribosyltransferase (ADPRT) activity. These two domains have numerous potential cellular targets, but the overall mechanism of ExoS action remains unclear. The effects of ExoS in a simple eukaryotic system, the yeast Saccharomyces cerevisiae, using a tetracycline-regulated expression system were studied. This system allowed controlled expression of ExoS in yeast, which was not possible using a galactose-induced system. ExoS was found to be an extremely potent inhibitor of yeast growth, and to be largely dependent on the activity of its ADPRT domain. ExoS produced a dramatic alteration in actin distribution, with the appearance of large aggregates of cortical actin, and thickened disorganized cables, entirely dependent on the ADPRT domain. This phenotype is suggestive of actin stabilization, which was verified by showing that the cortical aggregates of actin induced by ExoS were resistant to treatment with latrunculin A, an agent that prevents actin polymerization. ExoS increased the numbers of mating projections produced following growth arrest with mating pheromone, and prevented subsequent DNA replication, an effect that is again dependent on the ADPRT domain. Following pheromone removal, ExoS produced altered development of the mating projections, which became elongated with a swollen bud-like tip. These results suggest alternative pathways for ExoS action in eukaryotic cells that may result from activation of small GTPases, and this yeast expression system is well suited to explore these pathways.

ADP Ribose Transferases↗

PubMed1987

Interaction between the calcium and adenylate cyclase messenger systems in dispersed chief cells from guinea pig stomach. Possible cellular mechanism for potentiation of pepsinogen secretion.

To determine the role of the adenylate cyclase system in potentiation of enzyme secretion, we used cholera toxin to activate adenylate cyclase before examining the effects of agents on chief cell cAMP and pepsinogen secretion. Dispersed chief cells were obtained from guinea pig stomach by fractionation of mucosal cells on a Percoll gradient. Incubation of cells with 100 nM cholera toxin for 90 min and subsequent incubation with carbachol or cholecystokinin resulted in augmentation of cellular cAMP and potentiation of pepsinogen secretion. The rate of increase in cAMP with carbachol or cholecystokinin was similar to that for the potentiated secretory response. To determine the role of changes in cell calcium on these effects, we examined the actions of the ionophore A23187. In cells preincubated with cholera toxin, A23187 augmented cAMP and caused potentiation of pepsinogen secretion. The effects of A23187, carbachol, and cholecystokinin on cells preincubated with cholera toxin were abolished by removing extracellular calcium or by adding the calmodulin inhibitor trifluoperazine. These data indicate that in chief cells preincubated with cholera toxin, secretagogue-induced increases in cell calcium concentration activate calmodulin thereby augmenting levels of cAMP and causing potentiation of pepsinogen secretion. Modulation of adenylate cyclase by changes in chief cell calcium concentration appears to be one mechanism whereby secretagogue interaction can result in potentiation of pepsinogen secretion.

Adenylyl Cyclases↗

PubMed1997

Strong mucosal adjuvanticity of cholera toxin within lipid particles of a new multiple emulsion delivery system for oral immunization.

Cholera toxin (CT) is an effective mucosal adjuvant but causes significant intestinal secretion which limits its usefulness. In the present study we developed a new multiple emulsion (ME) delivery system into which antigen and CT could be incorporated and asked whether CT would retain its mucosal adjuvanticity when sequestered within emulsion particles. ME were selectively taken up into Peyer's patches, and those containing antigen plus CT generated intestinal secretory IgA and serum IgG antibody responses in mice comparable quantitatively and qualitatively to those occurring after oral immunization with soluble antigen plus CT. The ME particles containing CT did not cause intestinal secretion. The adjuvanticity of CT within ME was due to the CT present in the inner aqueous phase of the ME and was lost if CT binding was blocked by pre-incubation with GM1 ganglioside. Proteins incorporated in ME were protected from external acid, protease, and bile. We conclude that CT sequestered in ME, although unable to bind to the epithelium and thus stimulate intestinal secretion, still retains its mucosal adjuvanticity. Thus, the ability of CT to bind to enterocytes is not obligatory for its mucosal adjuvanticity.

Adjuvants, Immunologic↗