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PubMed1996

Cytolytic toxins from gram-negative bacteria.

Many Gram-negative bacterial pathogens synthesize cytolytic toxins as virulence factors. Most of these toxins generate pores in eukaryotic cell membranes, but there are apparently several different mechanisms of pore formation. Cytolysins of Gram-negative bacteria are usually synthesized as precursor proteins which are converted to the active toxins by modification or proteolytic processing. Such a requirement for activation is not common for cytolysins produced by Gram-positive bacteria. The extracellular secretion of cytolytic toxins from Gram-negative bacteria depends on specific transport systems.

Amino Acid Sequence↗

PubMed2001

Construction of mini-Tn5cyaA' and its utilization for the identification of genes encoding surface-exposed and secreted proteins in Bordetella bronchiseptica.

A mini-Tn5 transposon derivative, mini-Tn5cyaA', has been constructed. It contains a promoter-less and ribosome binding site-deficient reporter gene, encoding the catalytic domain of Bordetella pertussis adenylate cyclase toxin (CyaA'). We used this system to mutagenize B. bronchiseptica and we developed a screen for identification of mutants containing cyaA' translational fusions. This system was used to identify B. bronchiseptica genes that encode surface-exposed and secreted proteins.

Adenylate Cyclase Toxin↗

PubMed2001

Type III secretion systems and pathogenicity islands.

Some bacterial pathogens have evolved by acquiring pathogenicity islands (PIs), which are clusters of genes encoding virulence traits. PIs encoding the secretion of effector molecules via type III secretion (TTS) systems have been discovered in several gram-negative pathogens. TTS systems are involved in contact-dependent secretion of virulence factors and can facilitate delivery of toxins directly into target cells. The expanding list of bacteria found to contain clusters of TTS genes includes members of the genera Yersinia, Salmonella, Shigella, Escherichia, Pseudomonas, Bordetella, Burkholderia, Chlamydia and a number of plant pathogens or symbionts. This review discusses the current knowledge of the role of TTS PIs in pathogenicity, the genetic organisation and evolution of such systems,and the potential for using TTS systems as targets for novel treatments.

Bacterial Proteins↗

PubMed1994

Amylin (islet amyloid polypeptide) inhibition of insulin release in the perfused rat pancreas: implication of the adenylate cyclase/cAMP system.

Amylin inhibits glucose-induced insulin secretion in the rat pancreas. To study the mechanism by which amylin acts on the B-cell, we have investigated, in the perfused rat pancreas, the effect of synthetic rat amylin (75 pM) on insulin release elicited by secretagogues acting on the B-cell via the adenylate cyclase/cAMP system, i.e., glucagon (10 nM), gastric inhibitory polypeptide (GIP, 1 nM), forskolin (1 microM) and isobutylmethylxanthine (IBMX, 75 microM). In addition, we examined the effect of amylin on GIP-induced insulin release in pancreata from rats pretreated with pertussis toxin, an agent which inactivates certain Gi proteins coupled to adenylate cyclase. Amylin inhibited the insulin response to glucagon (approx. 70%), GIP (approx. 90%), IBMX (approx. 75%) as well as the early phase of forskolin-induced insulin output (approx. 74%). However, amylin failed to modify GIP-induced insulin release in pancreata obtained from pertussis toxin pretreated rats. These results would indicate that the inhibitory effect of amylin on insulin secretion could be, at least in part, attributed to its interfering with the adenylate cyclase/cAMP system. Furthermore, prevention of the inhibitory effect of amylin on GIP-induced insulin output by pertussis toxin pretreatment, supports the concept that amylin can inhibit insulin release via a pertussis toxin-sensitive Gi protein coupled to the adenylate cyclase system.

1-Methyl-3-isobutylxanthine↗

PubMed2002

A novel type II secretion system in Pseudomonas aeruginosa.

The genome sequence of Pseudomonas aeruginosa strain PAO1 has been determined to facilitate postgenomic studies aimed at understanding the capacity of adaptation of this ubiquitous opportunistic pathogen. P. aeruginosa produces toxins and hydrolytic enzymes that are secreted via the type II secretory pathway using the Xcp machinery or 'secreton'. In this study, we characterized a novel gene cluster, called hxc for homologous to xcp. Characterization of an hxcR mutant, grown in phosphate-limiting medium, revealed the absence of a 40 kDa protein found in the culture supernatant of wild-type or xcp derivative mutant strains. The protein corresponded to the alkaline phosphatase L-AP, renamed LapA, which is secreted in an xcp-independent but hxc-dependent manner. Finally, we showed that expression of the hxc gene cluster is under phosphate regulation. This is the first report of the existence of two functional type II secretory pathways within the same organism, which could be related to the high adaptation potential of P. aeruginosa.

Alkaline Phosphatase↗

PubMedDate not supplied

Secretion of virulence factors by Escherichia coli.

In order to interact with their host, pathogenic strains of E. coli need to secrete some virulence factors which can modify the metabolism of host cells, contributing to disease. Since E. coli is a Gram-negative bacteria, this secretion process involves the crossing of both the inner and the outer membranes. E. coli uses mainly four secretion mechanisms called type I, type II, type III and type IV secretion systems. In the type I secretion system, the secretion machinery is composed of three proteins forming a channel through the inner and outer membranes. It is a one-step mechanism. The secretion signal is present in the carboxyterminal region of the secreted protein but without proteolytic cleavage. In E. coli, the best studied type I secreted protein is haemolysin. In type II and type IV secretion systems, the crossing of the inner membrane involves the sec machinery with the cleavage of an aminoterminal signal sequence. The crossing of the outer membrane involves the formation of a pore either by other proteins (type II) or by the carboxyterminal region of the protein (type IV). The A-B toxins, such as heat labile enterotoxin, are secreted by the first mechanism and members of the IgA proteases are secreted by the second. The type III secretion system involves at least 20 proteins including cytoplasmic, inner membrane and outer membrane proteins. The originality of this system is the ability to inject secreted bacteria into the cytosol of the host cells. Such a system is found in attaching and effacing E. coli and in diffusely adhering E. coli.

Animals↗

PubMed1985

Alkaline phosphatases and adenylate cyclase in the normal and desensitized rat small intestine after acute cholera toxin challenge: a histochemical study.

The effect of cholera toxin (CT)-challenge on histochemically demonstrable activities of adenylatecyclase and alkaline phosphatase was investigated in rat small intestine, using an intestinal loop model. CT-challenge increased the activities of adenylatecyclase and alkaline phosphatases within 15 minutes, and the changes were confined to enterocytes in the upper third parts of the villi. There was no change in the staining of the crypt cells. There was an increased basal activity of both adenylatecyclase and alkaline phosphatases in animals desensitized to cholera toxin by multiple peroral exposures. CT-challenge in the desensitized rats did not further increase the enzyme activity. It is concluded that desensitization to secretagogues induces profound alterations in the cell systems responsible for fluid secretion.

Adenylyl Cyclases↗

PubMed1998

Characterization of the cvaA and cvi promoters of the colicin V export system: iron-dependent transcription of cvaA is modulated by downstream sequences.

Secretion of the Escherichia coli toxin colicin V was previously determined to be iron regulated via the Fur (ferric uptake regulator) protein, based on studies in fur mutants. The iron dependence of transcription and expression of cvaA, which encodes a transporter accessory protein, and cvi, encoding the colicin V immunity protein, was assessed under conditions of iron excess or depletion. Immunoblots showed that production of both Cvi and CvaA is iron dependent. The iron-dependent transcriptional start for cvaA identified by primer extension and S1 nuclease analysis, P1, lies 320 bp upstream of the translational start and is associated with a newly identified Fur binding site. Beta-galactosidase activity in transcriptional lacZ fusions with the P1 promoter alone is higher than with downstream sequences present and is induced 10-fold by iron depletion. Including immediate downstream regions with P1 enhances activity from P1 even more but reduces the induction by iron depletion fivefold. Including subsequent downstream sequences, however, down-modulates overall transcription from P1 almost fourfold. Deletion of a long stem-loop structure in this region alleviates the down-modulation by increasing transcription, indicating that the sequences or structure of this element may contribute to this down-regulation. Characterization of the cvi promoter by primer extension showed that it resides where predicted, about 50 bp upstream of cvi associated with a previously identified Fur binding site. The cvi promoter is also inducible by iron depletion. The modulating sequences from cvaA were placed downstream of the cvi promoter to test their effects in transcriptional fusions of the cvi promoter to lacZ. The fusion results showed that these sequences also modulate transcription of the cvi promoter in a manner similar to that of the cvaA promoter. The potential for up- and down-regulation within the long untranslated region downstream of the cvaA promoter suggests a novel mechanism that fine-tunes expression of the colicin V secretion genes.

ATP-Binding Cassette Transporters↗

PubMed2001

Modulation of host cell signalling by enteropathogenic and Shiga toxin-producing Escherichia coli.

The majority of Escherichia coli strains are harmless symbionts in the intestinal tract. However, there are several pathogenic forms, which are responsible for various diseases in humans and live stock. In this review we discuss the interactions between Shiga toxin-producing E. coli and enteropathogenic E. coli and their target host cells, describing their strategies to activate specific cellular signalling pathways which lead to subversion of critical physiological functions. We mainly concentrate on those pathogenic mechanisms that are dependent on a functional type III secretion system, but we also briefly discuss additional factors that contribute to the specific pathogenic profiles of Shiga toxin-producing E. coli and enreropathogenic E. coli.

Bacterial Proteins↗

PubMed2005

The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae.

Gram-negative bacteria use type II secretion systems for the transport of virulence factors and hydrolytic enzymes through the outer membrane. These sophisticated multi-protein complexes reach from the pore in the outer membrane via the pseudopilins in the periplasm and a multi-protein inner-membrane sub-complex, to an ATPase in the cytoplasm. The human pathogen Vibrio cholerae uses such a secretion machinery, called the Eps-system, for the export of its major virulence factor cholera toxin into the intestinal tract of the human host. Here, we describe the 2.4 A structure of the hetero-tetrameric complex of the N-terminal domain of the ATPase EpsE and the cytoplasmic domain of the inner membrane protein EpsL, which constitute the major cytoplasmic components of the Eps-system. A stable fragment of EpsE in complex with the cytoplasmic domain of EpsL was identified via limited proteolysis and facilitated the crystallization of the complex. This first structure of a complex between two different proteins of the type II secretion system reveals that the N-terminal domain of EpsE and the cytoplasmic domain of EpsL form a hetero-tetramer, in which EpsL is the central dimer and EpsE binds on the periphery. The dimer of EpsL in this complex is very similar to the dimer seen in the crystal structure of the native cytoplasmic domain of EpsL, suggesting a possible physiological relevance despite a relatively small 675 A2 buried solvent accessible surface. The N-terminal domain of EpsE, which forms a compact domain with an alpha+beta-fold, places its helix alpha2 in a mostly hydrophobic cleft between domains II and III of EpsL burying 1700 A2 solvent accessible surface. This extensive interface involves several residues whose hydrophobic or charged nature is well conserved and is therefore likely to be of general importance in type II secretion systems.

Amino Acid Sequence↗

PubMed2006

Influence of exogenous reproductive hormones on specific antibody production in genital secretions after vaginal vaccination with recombinant cholera toxin B subunit in humans.

The objective of this study was to investigate the influence of exogenous reproductive hormones on the local and systemic production of specific immunoglobulin A (IgA) and IgG antibodies after vaginal vaccination with recombinant cholera toxin subunit B (CTB). Three groups of women using either progesterone-containing intrauterine devices (n=9), oral contraceptives (n=8), or no hormonal contraceptive methods (n=9) were vaginally immunized twice, 2 weeks apart. Cervical secretions, vaginal fluids, and serum were collected before and after vaccination. Total and CTB-specific IgA and IgG antibodies in genital secretions and serum were analyzed by enzyme-linked immunosorbent assay. A majority of the women presented strong CTB-specific IgA and IgG antibody responses in cervicovaginal secretions after vaccination, whereas the antitoxin responses in serum were weaker. Exogenously administered steroid hormones did not seem to have any impact on the production of specific antibodies. Both the frequencies and the magnitudes of IgA and IgG antitoxin responses in genital secretions were comparable among the three immunization groups. An association, in particular for IgA, was found between the magnitudes of the CTB-specific antibody responses in cervical secretions and vaginal fluids after vaccination. The sensitivities and positive predictive values of vaginal antibody analyses to reflect responses in cervical secretions were also high, suggesting that vaginal fluids alone might be used for evaluation of genital immune responses in large-scale vaccination studies in the future.

Adult↗

PubMed2005

Triggered phagocytosis by Salmonella: bacterial molecular mimicry of RhoGTPase activation/deactivation.

Salmonella Typhimurium uses the type III secretion system encoded in the Salmonella pathogenicity island I (SPI-1 TTSS) to inject toxins (effector proteins) into host cells. Here, we focus on the functional mechanism of three of these toxins: SopE, SopE2, and SptP. All three effector proteins change the GTP/GDP loading state of RhoGTPases by transient interactions. SopE and SopE2 mimic eukaryotic G-nucleotide exchange factors and thereby activate RhoGTPase signaling pathways in infected host cells. In contrast, a domain of SptP inactivates RhoGTPases by mimicking the activity of eukaryotic GTPase-activating proteins. The Salmonella-host cell interaction provides an excellent example for the use of molecular mimicry by bacterial pathogens.

Animals↗

PubMed1998

alpha-Latrotoxin-induced quantal release of catecholamines from rat adrenal chromaffin cells.

alpha-Latrotoxin (alpha-LT) potently enhances quantal release of neurotransmitter from nerve terminals. To develop the adrenal chromaffin cell as a 'model' system for the study of mechanisms of toxin action, we used amperometry to examine secretion of catecholamines and spectrofluorometry to measure cytosolic Ca. Several key features of toxin action emerged. (1) Release occurs at concentrations of toxin >35 pM and the pattern of release changes from repeated brief bursts to more continuous discharges of varying duration as the toxin concentration increases. (2) Release requires extracellular calcium in the micromolar range, but not the activity of native voltage-dependent calcium channels. (3) Release is associated with a rise in cytosolic calcium to near micromolar range. (4) Provided calcium is later restored, release can be induced even though the toxin is applied and washed away in calcium-free saline. These features largely resemble those of alpha-LT action on nerve terminals. They suggest that in chromaffin cells, as in neurons, the Ca-dependence of toxin-enhanced quantal release is based on Ca entry through toxin-induced channels rather than a requirement of extracellular Ca for toxin binding.

Adrenal Glands↗

PubMed2005

XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus.

Pseudomonas aeruginosa is an opportunistic gram-negative pathogen equipped with multiple secretion systems. The type II secretion machinery (Xcp secreton) is involved in the release of toxins and enzymes. The Xcp secreton is a multiprotein complex, and most of its components share homology with proteins involved in type IV pili biogenesis. Among them, the XcpT-X pseudopilins possess characteristics of the major constituent of the type IV pili, the pilin PilA. We have shown previously that XcpT can be assembled in a multifibrillar structure that was called the pseudopilus. By using two different microscopic approaches, we show here that the pseudopili are preferentially isolated fibers rather than tight bundles. Moreover, none of the other four pseudopilins are able to form a pseudopilus, suggesting that the assembly of such a structure is a unique property of XcpT. Moreover, we show that 5 of the 12 Xcp proteins are not required for pseudopilus biogenesis, whereas they are for type II secretion. Most interestingly, we showed that one pseudopilin, XcpX, controls the assembly of XcpT into a pseudopilus. Indeed, when the number of XcpX subunits increases, the length of the pseudopilus decreases. Conversely, in the absence of XcpX, the pseudopilus length is abnormally long. Our results indicate that XcpT and XcpX directly interact with each other. Furthermore, this interaction induces a clear destabilization of XcpT. The interaction between XcpT and XcpX could be part of the molecular mechanism underlying the dynamic control of pseudopilus elongation, which could be crucial for type II-dependent protein secretion.

Bacterial Adhesion↗

PubMed2004

Pseudomonas aeruginosa causes acute lung injury via the catalytic activity of the patatin-like phospholipase domain of ExoU.

OBJECTIVE: Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU toxin being delivered directly into the eukaryotic cell cytosol through the type III secretion system. The amino-acid sequence of ExoU has a potato patatin-like phospholipase domain, similar to the sequence of mammalian Ca-independent phospholipase A2. We examined whether the acute lung injury caused by cytotoxic P. aeruginosa was dependent on the patatin-like phospholipase domain of ExoU. DESIGN: Laboratory investigation using an established mouse model for P. aeruginosa pneumonia with quantitative measurements of acute lung injury and mortality. SETTING: University experimental research laboratory. SUBJECTS: Balb/c mice. INTERVENTIONS: First, a site-directional mutation was introduced in the predicted catalytically active site of the patatin-like phospholipase domain of recombinant ExoU protein. The effect of the mutation on the catalytic activity of ExoU was tested by the in vitro lysophospholipase A assay. Second, the same site-directional mutation was introduced into the exoU gene of P. aeruginosa PA103. Mice were intratracheally infected with either a wild-type P. aeruginosa strain PA103 or an isogenic mutant containing the mutation in exoU. Acute epithelial lung injury, lung edema, bacteremia, and mortality were evaluated quantitatively. MEASUREMENTS AND MAIN RESULTS: Recombinant ExoU had lysophospholipase A activity. Site-directional mutations in the predicted catalytic site of ExoU caused a loss of the lysophospholipase A activity. Whereas the airspace instillation of PA103 caused acute lung injury and death of the infected mice, the airspace instillation of isogenic mutants secreting catalytically inactive ExoU were noncytotoxic and did not cause acute lung injury or death of the infected mice. CONCLUSION: Virulent P. aeruginosa causes acute lung injury and death by the cytotoxic activity derived from the patatin-like phospholipase domain of ExoU.

Acute Disease↗

PubMed2006

Leptin interferes with adrenocorticotropin/3',5'-cyclic adenosine monophosphate (cAMP) signaling, possibly through a Janus kinase 2-phosphatidylinositol 3-kinase/Akt-phosphodiesterase 3-cAMP pathway, to down-regulate cholesterol side-chain cleavage cytochrome P450 enzyme in human adrenocortical NCI-H295 cell line.

CONTEXT: Obesity has adverse effects on adrenocortical functions. Adipocyte-derived leptin, a biomarker molecule of obesity, may directly control adrenal steroidogenesis via an unclear mechanism. OBJECTIVE: We studied the mechanism underlying leptin action on adrenal steroidogenesis in human adrenocortical NCI-H295 tumor cell line. METHODS: Levels of progesterone, cortisol, and cAMP were determined by ELISA. Western blotting was used to detect protein amounts of P450 side-chain cleavage (P450scc), Janus kinase 2 (JAK2), Akt, and their phosphorylated forms. The mRNA expressions of P450scc and leptin receptors were measured by RT-PCR and real-time PCR. P450scc promoter activity was analyzed with a luciferase reporter system. RESULTS: Cholera toxin mimicked ACTH action by increasing adrenal cAMP levels and steroid secretion. Leptin did not affect basal release but significantly inhibited ACTH/cholera toxin-induced steroid secretion. The concomitant inhibitions by leptin on cholera toxin-induced protein and ACTH/cholera toxin-induced mRNA expression of P450scc were confirmed. Leptin inhibited ACTH/cholera toxin-induced CYP11A1 promoter activity via a known cAMP-responsive region located between -1.7 and -1.5 kb. Leptin activated phosphorylations of JAK2 and Akt. Inhibitory effects of leptin on ACTH/cholera toxin-induced cAMP levels, CYP11A1 promoter activity, and steroid secretion were blunted by either inhibitor of JAK2 (AG490) or phosphatidylinositol 3-kinase/Akt (wortmannin) as well as inhibitors of cAMP-degrading phosphodiesterases (PDEs), including nonspecific 3-isobutyl-1-methylxanthine and PDE3-specific SKF94836. Leptin failed to affect the inductions of CYP11A1 promoter activity and steroid secretion by PDE-nonhydrolyzable N(6)-monobutyryl-cAMP. CONCLUSIONS: Leptin interferes with ACTH/cAMP signaling, possibly through a cAMP-degrading mechanism involving activation of JAK2, phosphatidylinositol 3-kinase, and PDE3, to down-regulate P450scc expression and consequent adrenal steroidogenesis.

Adrenal Cortex↗

PubMed1990

Mutagenesis of Pseudomonas exotoxin in identification of sequences responsible for the animal toxicity.

Pseudomonas exotoxin (PE) is composed of three structural domains that are responsible for cell recognition, membrane translocation, and ADP-ribosylation. The deletion of the cell recognition domain (domain Ia) of PE results in a molecule that does not bind to target cells and has low toxicity in mice (Hwang, J., FitzGerald, D.J.P., Adhya, S., and Pastan, I. (1987) Cell 48, 129-136). To determine the specific sequences required for cell binding as well as cell and animal toxicity, a series of domain I mutants was constructed. Using a T7 promoter-based expression system and an OmpA signal sequence, large amounts of the various mutant toxins were secreted into the periplasm from which they were easily purified in milligram quantities. The data indicate that amino acids at positions 246, 247, and 249 have an important role in the toxicity of PE. Conversion of these amino acids to glutamic acid or glycine but not to lysine or deletion of amino acids 241-250 diminishes the toxicity of PE. When combined with a mutation at position 57 a molecule is created that has very low toxicity against cultured cells or in mice.

ADP Ribose Transferases↗

PubMed1997

Modulation of protein kinetics in chronic renal failure.

Many factors are involved in inducing muscle wasting and derangements of protein metabolism in chronic renal failure. Anorexia, low protein intake, hormonal abnormalities (increased cortisol and parathyroid hormone secretion, and insulin resistance), acidosis, abnormalities of the cytokine system, and other unidentified uremic toxins create a negative nitrogen balance and stimulate protein catabolism. The protein turnover rate (i.e., synthesis and degradation) is generally decreased in non-acidotic uremic patients. However, in uncorrected acidosis, protein degradation is accelerated and a rapid loss of body proteins supervenes. Nutrition can be improved in chronically uremic patients through pharmacological therapy that can control protein catabolism. Administration of growth hormone and insulin-like growth factor-1 has been shown to be effective. These hormones influence both glucose and glutamine metabolism. Another approach is the administration of pentoxifylline, which may have an anticatabolic effect by interfering with the tumor necrosis factor-alpha system.

Amino Acids↗