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PubMed2004

Analysis of subassemblies of pertussis toxin subunits in vivo and their interaction with the ptl transport apparatus.

Pertussis toxin (PT) has an AB(5) structure that is typical of many bacterial protein toxins; however, this toxin is more complex than many toxins since it is composed of five different subunit types, subunits S1 to S5. Little is known about how PT assembles in vivo and how and when it interacts with its secretion apparatus, known as the Ptl transporter. In order to better understand these events, we expressed subsets of the genes encoding the S1, S2, and/or S4 subunits of PT in strains of Bordetella pertussis that either did or did not produce the Ptl proteins. We found evidence to suggest that certain subassemblies of the toxin, including subassemblies consisting of the S1 subunit and incomplete forms of the B oligomer, can form in vivo, at least transiently. These results suggest that the B oligomer of the toxin does not need to completely form before interactions between the S1 subunit and B-oligomer subunits can occur in vivo. All subassemblies localized primarily to the membrane fraction of the cell. Moreover, we found that Ptl-mediated secretion occurs in a strain that produces S1 and an incomplete complement of B-oligomer subunits. These results indicate that subassemblies of the toxin consisting of the S1 subunit and a partial B oligomer can interact with the Ptl system.

Bacterial Proteins↗

PubMed1998

Enterocins L50A and L50B, two novel bacteriocins from Enterococcus faecium L50, are related to staphylococcal hemolysins.

Enterocin L50 (EntL50), initially referred to as pediocin L50 (L. M. Cintas, J. M. Rodríguez, M. F. Fernández, K. Sletten, I. F. Nes, P. E. Hernández, and H. Holo, Appl. Environ. Microbiol. 61:2643-2648, 1995), is a plasmid-encoded broad-spectrum bacteriocin produced by Enterococcus faecium L50. It has previously been purified from the culture supernatant and partly sequenced by Edman degradation. In the present work, the nucleotide sequence of the EntL50 locus was determined, and several putative open reading frames (ORFs) were identified. Unexpectedly, two ORFs were found to encode EntL50-like peptides. These peptides, termed enterocin L50A (EntL50A) and enterocin L50B (EntL50B), have 72% sequence identity and consist of 44 and 43 amino acids, respectively. Interestingly, a comparison of the deduced sequences of EntL50A and EntL50B with the corresponding sequences obtained by Edman degradation shows that these bacteriocins, in contrast to other peptide bacteriocins, are secreted without an N-terminal leader sequence or signal peptide. Expression in vivo and in vitro transcription/translation experiments demonstrated that entL50A and entL50B are the only genes required to obtain antimicrobial activity, strongly indicating that their bacteriocin products are not posttranslationally modified. Both bacteriocins possess antimicrobial activity on their own, with EntL50A being the most active. In addition, when the two bacteriocins were combined, a considerable synergism was observed, especially with some indicator strains. Even though the enterocins in some respects are similar to class II bacteriocins, several conserved features common to class II bacteriocins are absent from the EntL50 system. The enterocins have more in common with members of a small group of cytolytic peptides secreted by certain staphylococci. We therefore propose that the enterocins L50A and L50B and the staphylococcal cytolysins together constitute a new family of peptide toxins, unrelated to class II bacteriocins, which possess bactericidal and/or hemolytic activity.

Amino Acid Sequence↗

PubMed1995

Characterization of somatostatin receptor subtypes.

Somatostatin regulates endocrine and exocrine secretion, possesses antiproliferative properties and acts as a neurotransmitter/neuromodulator in the central nervous system. These effects are mediated by G protein-coupled receptors, of which at least five types have been cloned (sstr1-5). In radioligand-binding studies we have compared the binding properties of sstr1-5 with their activities as somatostatin receptors. All receptors identified so far bind somatostatin-14 and somatostatin-28 with high affinity. The similarities in receptor sequence and in the binding profiles of short synthetic somatostastin analogues such as octreotide, MK 678 or RC 160 for sstr1-5 indicate the existence of two classes of receptors sstr1/sstr4 with virtually no or very low affinity and sstr2/sstr3/sstr5 with intermediate to high affinity for the short somatostatin analogues. All five receptors mediate inhibition of adenylyl cyclase; this inhibition is sensitive to pertussis toxin. In vitro and in vivo studies suggest the importance of sstr2 and/or sstr5 in the inhibition of growth hormone release. The sstr2 receptor is apparently the predominant subtype expressed in somatostatin receptor-positive tumours. Evidence exists for the importance of sstr5 receptors in insulin secretion and sstr1 receptors in oncology. Somatostatin receptor-selective agonists and antagonists will help to explore new therapeutic opportunities in oncology as well as in endocrine and gastrointestinal disorders and those of the central nervous system.

Animals↗

PubMed2004

Inosine stimulates chemotaxis, Ca2+-transients and actin polymerization in immature human dendritic cells via a pertussis toxin-sensitive mechanism independent of adenosine receptors.

Inosine is an endogenous purine nucleoside, which is formed by adenosine deaminidase during adenosine breakdown and is released into the extracellular space from the sympathetic nervous system or injured cells. Here, we studied the biological activity of inosine on human dendritic cells (DC), which are specialized antigen presenting cells characterized by their ability to migrate from the blood to peripheral tissues, and then to secondary lymphoid organs where they initiate adaptive immune responses. In immature DC, inosine concentration-dependently stimulated Ca(2+)-transients, actin polymerization, and chemotaxis. Experiments with adenosine receptor antagonists and pertussis toxin (PTX) as well as desensitization studies suggested that the activity of inosine was mediated by a G protein-coupled receptor pathway independent of adenosine receptors. DC, induced to mature by lipopolysaccharide, lost their ability to respond towards inosine with these activities. Moreover, inosine did neither influence membrane expression of CD54, CD80, CD83, CD86, HLA-DR, and MHC class I molecules nor modulated secretion of interleukin (IL)-12, IL-10, and tumor necrosis factor alpha in immature and lipopolysaccharide-matured DC. In aggregate, our study indicates that inosine may be involved in the trafficking control system of immature DC, and mediates its chemotactic activity by a PTX-sensitive mechanism independent of adenosine receptors.

Actins↗

PubMed1998

Local and systemic responses against ricin toxin promoted by toxoid or peptide vaccines alone or in liposomal formulations.

The objective of this study was to develop a vaccine which would ultimately protect man from the lethal effects of inhaled ricin toxin. Porton rats have previously been protected from lethal quantities of inhaled ricin by subcutaneous (s.c.) ricin toxoid vaccine, but not without lung damage. This situation might be improved by an alternative vaccine such as the A chain of ricin, already known to protect against inhaled ricin. Another option would be to improve respiratory tract immunity by local vaccination in conjunction with liposomal formulation with a view to enhancing lung secretion of immune IgA. While boosted s.c. doses of ricin toxoid or A chain produced indistinguishable systemic immune responses 3 weeks later, when delivered by the intratracheal (i.t.) route, the A chain failed to elicit a specific immune response, unlike ricin toxoid. This situation was overcome by liposomal formulations and although ricin toxoid was readily encapsulated in liposomes, A chain was not. However, by simply mixing A chain and liposomes in the same weight ratio determined for liposomal toxoid, systemic immune responses for each formulation were indistinguishable 1 week after boosting. Ricin antibody responses in lung fluid monitored 1, 3, 7 and 14 days after i.t. challenge with ricin were statistically indistinguishable, but the group vaccinated with liposomal toxoid secreted 28.7% IgA compared with 0.9-14.9% for the A chain liposomal group. From this, it might be anticipated that the lungs would be better protected by liposomally-encapsulated ricin toxoid than by the A chain-liposome mixture.

Animals↗

PubMed1998

Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli has been responsible for an increasing number of large food-borne outbreaks of bloody diarrhea and hemolytic uremic syndrome. Recent developments in our understanding of the pathogenesis of disease due to enterhemorrhagic E. coli include the description of a pathogenicity island, a type III secretion system and potential plasmid-encoded virulence factors. Recent developments in our understanding of the epidemiology include a recognition of a widening spectrum of vehicles.

Animals↗

PubMed2003

Gram-negative bacterial ATP-binding cassette protein exporter family and diverse secretory proteins.

Protein translocation to the extracellular space is essential for the invasion, colonization, and survival of pathogenic gram-negative bacteria within a host organism. In addition to the N-terminal signal sequence-dependent secretion system, which is specific for protein transport to the periplasmic space, there are five major systems (type I, II, III, IV, and V) that are known to be involved in protein secretion into the extracellular space. Of the systems, the type I pathway, which is composed of three membrane components including an ATP-binding cassette (ABC) protein, translocates proteins into the extracellular space from the cytosol by directly using the energy generated from ATP hydrolysis, and therefore, the system is a member of the ABC transporter family and is also known as the ABC exporter. To date, ABC exporters have been discovered to be involved in the secretion of a wide variety of exoproteins including RTX (repeats-in-toxin) toxins, cell surface layer proteins, proteases, lipases, bacteriocins, heme-acquisition proteins, and nodulation-related proteins such as the exoglucanases of gram-negative bacteria. A secretory protein and its associated specific ABC exporter are encoded in the same gene cluster in most cases, and ABC exporters show substrate specificity for secretion. Consequently, ABC exporters are present based primarily on the number of secretory protein genes. A secretion signal is situated in the C-terminal region of secretory proteins, however, the characteristics of the secretion signal are not fully understood. Secretory substrates and their linked ABC exporters are reviewed in the following paper.

Journal Article↗

PubMed1994

Agents that promote protein phosphorylation inhibit the activity of the Na+/Ca2+ exchanger and prolong Ca2+ transients in bovine chromaffin cells.

The Na+/Ca2+ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na+/Ca2+ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na+/Ca2+ exchanger of these cells. Cells treated with 1 mM dibutyryl cyclic AMP (dbcAMP), 1 microM phorbol 12,13-dibutyrate, 1 microM okadaic acid, or 100 nM calyculin A showed lowered Na+/Ca2+ exchange activity and prolonged cytosolic Ca2+ transients caused by depolarization. A combination of 10 nM okadaic acid and 1 microM dbcAMP synergistically inhibited Na+/Ca2+ exchange activity. Conversely, 50 microM 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na+/Ca2+ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na+/Ca2+ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na+/Ca2+ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.

Animals↗

PubMed1993

Aeromonas spp. can secrete Escherichia coli alkaline phosphatase into the culture supernatant, and its release requires a functional general secretion pathway.

Aerolysin is a channel-forming protein secreted by Aeromonas hydrophila. To determine if regions of aerolysin could direct the secretion of another protein, portions of aerA were fused to phoA, the Escherichia coli alkaline phosphatase gene and cloned into E. coli, Aeromonas salmonicida, and A. hydrophila. We were surprised to find that secretion of the enzyme by both Aeromonas spp. was independent of the aerolysin segments fused to it. The smallest fusion product contained only the signal sequence and two amino acids of aerolysin. The largest had more than 90% of the aerolysin molecule. The fusion proteins were found in the periplasms of E. coli and A. salmonicida grown in LB medium containing glucose, as well as in the shocked cells. Aerolysin itself was secreted by A. salmonicida under these conditions. In contrast, when A. salmonicida containing any of the fused genes was grown in LB medium without glucose, most of the alkaline phosphatase activity was extracellular, whereas beta-lactamase remained in its normal periplasmic location. Similar results were obtained with A. hydrophila. The change in location of the enzyme in A. salmonicida appeared to be related to the pH of the growth medium. A. salmonicida and A. hydrophila also secreted native E. coli alkaline phosphatase, but A. hydrophila strains with mutations in the general secretion pathway were unable to release the enzyme. We conclude that the Aeromonas secretion system can recognize the E. coli enzyme as an extracellular protein and direct it outside the cell.

Aeromonas↗

PubMed2003

c-Jun NH2-terminal kinase-mediated signaling is essential for Pseudomonas aeruginosa ExoS-induced apoptosis.

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa mainly affects immunocompromised individuals as well as patients with cystic fibrosis. In a previous study, we showed that ExoS of P. aeruginosa, when injected into host cells through a type III secretion apparatus, functions as an effector molecule to trigger apoptosis in various tissue culture cells. Here, we show that injection of the ExoS into HeLa cells activates c-Jun NH(2)-terminal kinase (JNK) phosphorylation while shutting down ERK1/2 and p38 phosphorylation. Inhibiting JNK activation by expression of a dominant negative JNK1 or with a specific JNK inhibitor abolishes ExoS-triggered apoptosis, demonstrating the requirement for JNK-mediated signaling. Following JNK phosphorylation, cytochrome c is released into the cytosol, leading to the activation of caspase 9 and eventually caspase 3. Although c-Jun phosphorylation is also observed as a result of JNK activation, ongoing host protein synthesis is not essential for the apoptotic induction, suggesting that c-Jun- or other AP-1-driven activation of gene expression is dispensable in this process. Therefore, ExoS has opposing effects on different cellular pathways that regulate apoptosis: it shuts down host cell survival signal pathways by inhibiting ERK1/2 and p38 activation, and it activates proapoptotic pathways through activation of JNK1/2 leading ultimately to cytochrome c release and activation of caspases. These results highlight the modulation of host cell signaling by the type III secretion system during interaction between P. aeruginosa and host cells.

ADP Ribose Transferases↗

PubMed1999

Cell adhesion molecules in the pathogenesis of and host defence against microbial infection.

Eukaryotic cell adhesion molecules (CAMs) are used by various cells and extracellular molecules in host defence against infection. They are involved in many processes including recognition by circulating phagocytes of a site of inflammation, transmigration through the endothelial barrier, diapedesis through basement membrane and extracellular matrix, and release of effector mechanisms at the infected site. CAMs involved in leucocyte-endothelial cell interaction include the selectins, integrins, and members of the immunoglobulin superfamily. However, CAMs are also used by various microorganisms (protozoa, fungi, bacteria, and viruses) during their pathogenesis. For example, bacteria that utilise CAMs include Mycobacterium tuberculosis, Listeria monocytogenes, Yersinia spp, enteropathogenic Escherichia coli, Shigella spp, Neisseria spp, Bordetella spp, and Borrelia burgdorferi. In addition, CAMs are involved in the pathogenetic effects of the RTX toxins of Pasteurella haemolytica, Actinobacillus actinomycetemcomitans, and the superantigen exotoxins of Staphylococcus aureus and Streptococcus pyogenes. A recurrent and topical theme of potential importance within the bacterial group is the intimate relation between CAMs, bacterial protein receptors, and type III secretion systems. For example, the IpaBCD protein complex is secreted by the type III system of Shigella flexneri and interacts with alpha 5 beta 1 integrin on the eukaryotic cell surface, followed by Rho mediated internalisation; this illustrates the relevance of cellular microbiology. CAMs might prove to be novel therapeutic targets. Comparative genomics has provided the knowledge of shared virulence determinants among diverse bacterial genera, and will continue to deepen our understanding of microbial pathogenesis, particularly in the context of the interaction of prokaryotic and eukaryotic molecules.

Cell Adhesion Molecules↗

PubMed2005

[Impact of Ph-SA on the expression of delta-toxin in Staphylococcus aureus].

OBJECTIVE: We carried out this pilot study in an attempt to observe the effect of Ph-SA on the secretion of delta-toxin in S. aureus ATCC29213. METHODS: When A595 reaching about 0. 2, the bacteria were divided into three groups and were treated by PBS, penicillin and Ph-SA respectively. The amount of delta-toxin from different groups and growth phases was analyzed by high performance liquid chromatography. RESULTS: It was observed that the secretion of delta-toxin in Ph-SA group was less than that in the other two groups at the same time (P < 0.05). CONCLUSION: We postulate that Ph-SA has the negative effect on the activity of agr system, acting as a pseudo-ligand.

Bacterial Proteins↗

PubMed1983

Escherichia coli heat-stable enterotoxin: biochemical and physiological effects on the intestine.

E. coli which elaborate suckling mouse active small MW heat-stable enterotoxin (STa), are important causes of diarrhea in animals and man. These STa's share the property of causing intestinal secretion and diarrhea by virtue of inhibiting the absorption of sodium and chloride and possibly stimulating the secretion of chloride. STa's seem to act in the colon as well as the small intestine and the alterations in intestinal ion and water transport are probably mediated by the guanylate cyclase-cyclic GMP system. Glucose transport is unaffected. STa also causes alterations in the myoelectrical activity of the small intestine which may result in the loss of normal peristaltic activity. STa binds in a reversible fashion to specific receptors on the surface of small intestinal and colonic epithelial cells. The mechanisms whereby occupation of the STa receptors lead to activation of the guanylate cyclase system and intestinal secretion are unknown but may involve influx of calcium through calcium channels, stimulation of prostaglandin synthesis and release of free radicals.

Action Potentials↗

PubMedDate not supplied

Induction of an enteric Ig-response against ovalbumin and stimulation of the response by cholera toxin and its B-subunit in mice.

We induced ovalbumin (OA)-specific antibody responses in the murine small intestine and quantitated the responses by counting ovalbumin-specific antibody-containing cells (OACC) in the intestinal lamina propria, and by measuring anti-OA Ig titers in intestinal secretions. OA is a "weak" mucosal antigen and we could only induce intestinal anti-OA responses by intraperitoneal, primary injection of OA in water-in-oil emulsion and a mucosal booster with OA. Double staining of OACC for OA- and isotype-specificity demonstrated that 95% of all mucosal (intestinal) OACC produced IgA, whereas 95% of all systemic (splenic) OACC produced IgG. The intestinal anti-OA response could be stimulated by addition of cholera toxin (CT) or cholera toxin B-subunit (CTB) during the mucosal booster immunization. The stimulatory effect of CT did not depend on covalent coupling of CT and OA, while the stimulatory effect of CTB required covalent coupling of CTB with OA. Mucosal (intraduodenal) application of OA as such does not prime for a mucosal and systemic OACC response but induces tolerance. We demonstrated that coupling of OA with CT or CTB could overcome the inability of mucosal application of OA to prime for a mucosal OACC response. Although CT proved to be much more effective than CTB in stimulation of mucosal immune responses and possibly in prevention of tolerance induction, our results indicate that CTB is a useful (and safer) alternative.

Animals↗

PubMedDate not supplied

'Depletion' of ATP by 2-deoxyglucose: secretion by electroporated human neutrophils is not restored by readdition of ATP.

Studies of intracellular signal transduction are facilitated by the use of permeabilized cell systems, which permit the ready manipulation of the cytosol. These model systems have helped to define the roles that small solutes, particularly Ca2+ and nucleotides, play in stimulus-response coupling. In circumstances where the full depletion of intracellular ATP contents is required, some investigators have resorted to prior treatment with metabolic toxins, with the expectation that the role of ATP in signal transduction could then be more unambiguously studied. However, in the work reported here, we found that treatment with 2-deoxyglucose (2-DOG) irreversibly altered the cells: when poisoned human neutrophils were then permeabilized, the cells failed to degranulate well in response to Ca2+, and their sensitivity to Ca2+ could not be recovered by the readdition of ATP. Inhibition of secretion by 2-DOG was most pronounced when low concentrations of Ca2+ were used as the stimulus. Preincubation of the cells with only 1 mM 2-DOG for 10 min at 37 degrees C (prior to washing and permeabilizing the cells) was sufficient for maximal inhibition. Even without preincubation, high concentrations of 2-DOG directly inhibited secretion. The refractory nature of poisoned cells was not restored by the presence of Mg2+ and/or ATP. The protein kinase C agonist phorbol myristate acetate also did not restore sensitivity of secretion to Ca2+. Addition of ATP and/or GTP to the permeabilization medium (to maximize penetration of the nucleotides) failed to restore sensitivity; tracer studies demonstrated that these conditions were adequate for repletion of the nucleotide pool. These data indicate that human neutrophils poisoned with 2-DOG were irreversibly altered, such that restoration of the putative deficiency (ATP) was without effect. Experiments in which such preincubation measures are employed should be viewed with caution.

Adenosine Triphosphate↗

PubMed1992

Comparative effects of atrial natriuretic peptide and E. coli heat-stable toxin on rat intestinal transport.

Conflicting data have been published in favor of or against a secretory effect of atrial natriuretic peptide (ANP) in the intestine. The reported effects resemble that of Escherichia coli heat-stable enterotoxin (ST). In this work the effects of ANP were studied in well established experimental systems and compared with that of ST. Both peptides induced a prompt secretion of water, Na, and Cl with no effects on K net transport in the in vivo rat perfused jejunum. The addition of ST, but not of ANP, evoked an increase of short circuit current in rat intestinal mucosa mounted in Ussing chambers. ST induced a significant increase in guanylate cyclase activity in intestinal homogenates, whereas ANP showed no effect. No binding sites for ANP were detected in basolateral or brush border membranes, nor in isolated enterocytes by a suction filtration technique. In conclusion, ANP acts as a short-lived intestinal secretagogue in the rat. Its mechanism of action is different from that of E. coli ST and appears to be indirect, since is not mediated by specific intestinal receptors and is not evident in vitro.

Animals↗

PubMed2006

Construction of a Vibrio splendidus mutant lacking the metalloprotease gene vsm by use of a novel counterselectable suicide vector.

Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the PBAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.

Animals↗

PubMed2003

Role of Rho in rabbit parietal cell.

Rho is known as an important regulator of actin microfilament formation. We were led to study it because a dynamic rearrangement of actin filaments occurs during activation of gastric acid secretion. In order to use specific probes, the rabbit gastric gland culture system was employed and the various genes were expressed using adenovirus vector. When the constitutive active mutant of Rho (RhoAV14) was expressed, histamine- or carbachol-stimulated acid secretion monitored by (14)C-aminopyrine accumulation was inhibited. Conversely, expression of C3 toxin, the specific inhibitor of Rho, and expression of G(12/13)-specific regulator of G-protein signaling domain, the specific inhibitor of G(12/13) which is considered to be an upstream mediator of Rho, both potentiated acid secretion stimulated by the agonists. F-actin staining of parietal cell expressing RhoAV14 revealed that the microfilament supporting the intracellular canaliculi (not on the basolateral membrane) almost disappeared. No clear changes in the intracellular localization of Rho were observed during stimulation of parietal cell. In resting glands, the endogenous active form of Rho was relatively high, and it decreased during histamine stimulation. The finding that any treatment which inhibit Rho augment acid secretion whereas those that activate Rho inhibit secretion strongly suggests that the Rho-pathway conducts a negatively regulating signal in parietal cell activation, possibly via site-specific regulation of actin microfilaments.

Actin Cytoskeleton↗