Escherichia coli CdtB mediates cytolethal distending toxin cell cycle arrest.

We previously reported that the CdtB polypeptide of Escherichia coli cytolethal distending toxin (CDT) shares significant pattern-specific homology with mammalian type I DNases. In addition, the DNase-related residues of CdtB are required for cellular toxicity. Here we demonstrate that purified CdtB converts supercoiled plasmid DNA to relaxed and linear forms and promotes cell cycle arrest when combined with an E. coli extract containing CdtA and CdtC. CdtB alone had no effect on HeLa cells, however; introduction of the polypeptide into HeLa cells by electroporation resulted in cellular distension, chromatin fragmentation, and cell cycle arrest, all of which are consequences of CDT action. In contrast to these findings, purified CdtB(H154A) lacked both DNA-nicking and cell cycle arrest activities. These results suggest a functional relationship between DNase-related residues in CdtB and CDT biological activity.

DNase I homologous residues in CdtB are critical for cytolethal distending toxin-mediated cell cycle arrest.

Cytolethal distending toxins (CDTs) block cell division by arresting the eukaryotic cell cycle at G2/M. Although previously not recognized in standard BLAST searches, a position-specific iterated (PSI) BLAST search of the protein data bank using CDT polypeptides as query sequences indicated that CdtB bears significant position-specific homology to type I mammalian DNases. The PSIBLAST sequence alignment reveals that residues of DNase I involved in phosphodiester bond hydrolysis (His134 and His252) are conserved in CdtB as well as their respective hydrogen bond pairs (Glu78 and Asp212). CdtB also contains a pentapeptide motif found in all DNase I enzymes. Further, crude CDT preparations possess detectable DNase activity not associated with identical preparations from control cells. Five CdtB mutations in amino acids corresponding to DNase I active site residues were prepared and expressed together with wild-type CdtA and CdtC polypeptides. Mutation in four of the five DNase-specific active site residues resulted in CDT preparations that lacked DNase activity and failed to induce cellular distension or arrest division of HeLa cells. The fifth mutation, Glu86 (Glu78 in DNase I), retained the ability to induce a moderate level of cell cycle arrest and displayed reduced DNase activity relative to wild-type CDT. Together, these data suggest that the CDT holotoxin has intrinsic DNase activity that is associated with the CdtB polypeptide and that this DNase activity may be responsible for the CDT-induced cell cycle arrest.

Interaction of Escherichia coli heat-stable enterotoxin B with rat intestinal epithelial cells and membrane lipids.

The binding of 125I-labeled Escherichia coli heat-stable enterotoxin B to rat intestinal epithelial cells was unsaturable and nonspecific, at concentrations well above that required to mediate biological events. Following its interaction with intestinal cells, approximately 50-80% of heat-stable enterotoxin B remained stably associated with the cells, implying that it was partitioned into the membrane and/or internalized by the cell. The toxin bound with different affinities to lipids isolated from intestinal epithelial cells, phospholipids, glycolipids, neutral lipids and to model membrane vesicles containing negatively charged lipids. These results indicate that heat-stable enterotoxin B utilizes the membrane bilayer, rather than a surface protein or glycoprotein in modulating toxin-induced enterotoxicity.

Effect of cytolethal distending toxin on F-actin assembly and cell division in Chinese hamster ovary cells.

Cytolethal distending toxin (CDT) is a newly described toxin produced by a number of enteropathogens, including Campylobacter jejuni, various Escherichia coli strains, and a few Shigella species. CDT induces distension and eventual death of a number of transformed cell lines. Here, we extend previous studies by demonstrating that morphological changes in CDT-treated Chinese hamster ovary cells are coincident with changes in cytoskeletal structure and an inhibition of cell proliferation. CDT-treated cells underwent a progressive accumulation of F-actin assemblies which microscopically resembled actin stress fibers. Accumulation of the stress fiber-like structures in CDT-treated cells was accompanied by an apparent blockage of cell division. Multinucleation was detected in some cells but did not constitute a significant feature of CDT action. Although toxin-treated cells failed to divide, cell viability remained high for the first 4 days following toxin treatment, as evidenced by trypan blue exclusion and neutral red uptake. [3H]thymidine incorporation studies on CDT-treated cells were consistent with a blockage of cell proliferation without a direct inhibition of DNA synthesis. Although the progression of toxin action developed slowly, a 2-min exposure to CDT resulted in an irreversible development of toxicity. Together, our data indicate that CDT affects F-actin assembly within target cells and may interrupt the regulation or function of cell cycle-dependent events leading to cytokinesis.

Interaction of Escherichia coli heat-stable enterotoxin B with cultured human intestinal epithelial cells.

Binding of Escherichia coli heat-stable enterotoxin B (STb) to the human intestinal epithelial cell lines T84 and HT29 and to polarized T84 cells was studied to define the initial interaction of this peptide toxin with target cells. Equilibrium and competitive binding isotherms showed that 125I-STb bound specifically to T84 and HT29 cells; however, the toxin-epithelial cell interactions could be characterized by low-affinity binding (< or = 10(5) M(-1)) to a high number of binding sites (> or = 10(6) per cell). STb binding to T84 and HT29 cells as a function of 125I-STb concentration did not approach saturation at levels well above the effective biological concentration of STb for fluid secretion. Treatment of the 125I-STb-bound T84 and HT29 cells with an acidic saline solution to remove surface-bound toxin revealed that only approximately 55% +/- 10% of 125I-STb could be removed by this treatment at 4 degrees C, suggesting that approximately half of the bound STb was stably associated with the plasma membrane and/or internalized into the cytoplasm. Similar results were obtained when binding and internalization experiments were conducted at 22 and 37 degrees C. Immunofluorescence studies demonstrated that the strongest signal for STb appeared in the plasma membrane even after acid treatment. Toxin-treated cells also displayed diffuse cytoplasmic staining, indicating that once cell bound, STb did not appear to preferentially associate with membrane vesicles or cellular organelles. Binding and subsequent internalization of 125I-STb were not affected by treatment of the cells with trypsin, endoglycosidase F/peptide N-glycosidase F, Vibrio cholerae neuraminidase, tunicamycin, or 5 mM sodium chlorate, which blocks sulfation of surface proteoglycans. In addition, the internalization process was not altered by preincubation of the cells with the cytoskeleton inhibitors cytochalasin D and colchicine or cellular perturbants (i.e., 0.45 M sucrose and 5 mM sodium azide), indicating that cell surface proteins or carbohydrates did not function as STb receptors. The binding of 125I-STb to polarized T84 cells was also examined, and the total and nonspecific binding isotherms were found to overlap, indicating that the apical surface of polarized T84 cells did not contain a specific receptor for STb. In comparison to undifferentiated cells, twice the amount of bound STb (approximately 80% +/- 10%) was removable from polarized T84 cells after treatment with acidic solution. The percentage of surface-bound STb to polarized T84 cells did not vary significantly with the transepithelial electrical resistance of the cells or when STb was applied basolaterally. Together, our results indicate that STb binds with relatively low affinity to the plasma membrane of cultured intestinal epithelial cells and polarized T84 cells, probably to membrane lipids, and becomes stably associated with the lipid bilayer. The fact that a significant portion of the bound STb becomes free in the cytoplasm, even at a low temperature, suggests that the bound toxin may directly traverse the membrane bilayer.

Judging relative duration: the role of rule and instructional variables.

Humans compared 2 durations according to different rules. Some judged which duration lasted longer, some judged whether the duration ratio was less or greater than 3:1, and others judged according to a same-different rule. Under each condition, 1/2 of the participants had advance knowledge of the rule, whereas the others acquired the discrimination solely on the basis of informative feedback. Discrimination was affected by both factors. Same-different and ratio comparisons were less accurate than ordinal comparisons. Rule knowledge affected the ratio and same-different comparisons but did not affect judgment about which duration lasted longer. Debriefing of uninformed participants revealed that most guessed that the rule involved judging which duration lasted longer. These results highlight the role of linguistic variables in humans' relational comparisons of duration.

Release of serotonin from RBL-2H3 cells by the Escherichia coli peptide toxin STb.

Heat-stable enterotoxin b (STb) of Escherichia coli is a 48-amino acid basic, disulfide-bonded peptide that causes intestinal secretion in experimental animal models. Recent evidence suggests that the in vivo mechanism of STb action involves release of 5-hydroxytryptamine (5-HT) and production of prostaglandin E2 (PGE2). Here we show STb-mediated release of 5-HT from rat basophilic leukemic cells (RBL-2H3), a mast cell line model used extensively to study 5-HT release. Increasing concentrations of biologically active STb resulted in a dose-dependent release of 5-HT from RBL-2H3 cells. In contrast to these results, reduced and alkylated STb had no effect on 5-HT release. Release of 5-HT from RBL-2H3 cells was independent of extracellular calcium ions and did not involve changes in the intracellular concentration of free Ca2+. In addition, pertussis toxin treatment completely blocked 5-HT release, indicating a role for a pertussis toxin-sensitive G-protein in the mechanism of 5-HT release from this cell type.

Contribution of individual disulfide bonds to biological action of Escherichia coli heat-stable enterotoxin B.

Heat-stable enterotoxins (STs) of Escherichia coli are peptides which alter normal gut physiology by stimulating the loss of water and electrolytes. The action of heat-stable toxin B (STb) is associated with an increase in levels of lumenal 5-hydroxytryptamine and prostaglandin E2, known mediators of intestinal secretion. In addition, the toxin is responsible for elevation of cytosolic calcium ion levels in cultured cells. STb is a 48-amino-acid basic peptide containing four cysteine residues and two disulfide bonds. Previous work indicates that disulfide bonds are required for intestinal secretory activity, and yet the relative contribution of the two bonds to toxin stability and action is presently unclear. Site-directed mutagenesis was used to alter the cysteine residues of STb to assess the role of the individual disulfide bonds in toxin activity. Our results indicate that loss of a single disulfide bond was sufficient to abolish the intestinal secretory and G protein-coupled calcium ion influx activities associated with STb toxicity. Loss of toxin action was not a function of increased sensitivity of STb mutants to proteolysis, since mutant toxins displayed proteolytic decay rates equivalent to that of wild-type STb. Circular dichroism spectroscopy of mutant STb toxins indicated that single-disulfide-bond elimination did not apparently affect the toxin secondary structure of one mutant, STbC33S,C71S. In contrast, the alpha-helical content of the other disulfide bond mutant, STbC44S,C59G, was significantly altered, as was that of reduced and alkylated authentic STb. Since both Cys-Cys mutant STbs were completely nontoxic, the absence of biological activity cannot be explained by dramatic secondary structural changes alone; keys to the conformational requirements for STb toxicity undoubtedly reside in the three-dimensional structure of this peptide.

The structure of Escherichia coli heat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism.

The heat-stable enterotoxin b (STb) is secreted by enterotoxigenic Escherichia coli that cause secretory diarrhea in animals and humans. It is a 48-amino acid peptide containing two disulfide bridges, between residues 10 and 48 and 21 and 36, which are crucial for its biological activity. Here, we report the solution structure of STb determined by two- and three-dimensional NMR methods. Approximate interproton distances derived from NOE data were used to construct structures of STb using distance-geometry and simulated annealing procedures. The NMR-derived structure shows that STb is helical between residues 10 and 22 and residues 38 and 44. The helical structure in the region 10-22 is amphipathic and exposes several polar residues to the solvent, some of which have been shown to be important in determining the toxicity of STb. The hydrophobic residues on the opposite face of this helix make contacts with the hydrophobic residues of the C-terminal helix. The loop region between residues 21 and 36 has another cluster of hydrophobic residues and exposes Arg 29 and Asp 30, which have been shown to be important for intestinal secretory activity. CD studies show that reduction of disulfide bridges results in a dramatic loss of structure, which correlates with loss of function. Reduced STb adopts a predominantly random-coil conformation. Chromatographic measurements of concentrations of native, fully reduced, and single-disulfide species in equilibrium mixtures of STb in redox buffers indicate that the formation of the two disulfide bonds in STb is only moderately cooperative. Similar measurements in the presence of 8 M urea suggest that the native secondary structure significantly stabilizes the disulfide bonds.

Involvement of 5-hydroxytryptamine and prostaglandin E2 in the intestinal secretory action of Escherichia coli heat-stable enterotoxin B.

The intestinal secretory action of Escherichia coli heat-stable enterotoxin B (STb) is poorly defined. Previous work indicates that STb causes loss of intestinal fluid and electrolytes by a mechanism independent of elevated levels of cyclic nucleotides, the hallmark of other E. coli cytotonic enterotoxins. In the work described in this report, we observed that treatment of ligated rat intestinal loops with purified STb of E. coli resulted in a dose-dependent rise in intestinal secretion concomitant with dose-related increases in levels of serotonin (5-hydroxytryptamine [5-HT]) and prostaglandin E2 (PGE2). Treatment of rats with the 5-HT2 receptor antagonist ketanserin prior to STb challenge resulted in significant (P < 0.05) reduction in intestinal secretion. Blockage of 5-HT2 receptors with ketanserin also reduced (P < 0.05) the level of PGE2 observed following STb treatment, indicating that at least a portion of the PGE2 was formed in response to 5-HT2 receptor stimulation. In a similar fashion, indomethacin, an inhibitor of cyclooxygenase activity, significantly reduced the level of secretion (P < 0.05) observed following STb treatment yet had no effect on 5-HT levels. Treatment of rats with both ketanserin and indomethacin further reduced STb-mediated secretion to a level not attained by either drug alone. Taken together, our data suggest that secretion due to STb involves both 5-HT and PGE2 as intestinal secretagogues. Furthermore, PGE2 formation appears to arise through both 5-HT-dependent and 5-HT-independent pathways.

Duration comparison: relative stimulus differences stimulus age, and stimulus predictiveness.

Under a psychophysical trials procedure, pigeons were presented with a red light of one duration followed by a green light of a second duration. Eight geometrically spaced base durations were paired with one of four shorter and four longer durations as the alternate member of a duration pair, with different pairs randomly intermixed. One choice was reinforced if red had lasted longer than green, and a second choice was reinforced if green had lasted longer. Performance was compared when all the base durations and their pair members were included (entire-range condition) or when only the four longest base durations and their comparison durations (restricted-range condition) were used. Discrimination sensitivity decreased for longer duration pairs under both conditions, supporting a memory-based account. Sensitivity was lower under the restricted-range condition. Under both conditions, a bias to report "green as longer" increased as the second green duration increased. Bias changed as a matching function of the green-duration predictiveness of the correct choice. The results are related to a quantitative model of timing and remembering proposed by Staddon.

Calcium influx mediated by the Escherichia coli heat-stable enterotoxin B (STB).

The heat-stable enterotoxin B (STB) of Escherichia coli is a 48-amino acid extracellular peptide that induces rapid fluid accumulation in animal intestinal models. Unlike other E. coli enterotoxins that elicit cAMP or cGMP responses in the gut [heat-labile toxin (LT) and heat-stable toxin A (STA), respectively], STB induces fluid loss by an undefined mechanism that is independent of cyclic nucleotide elevation. Here we studied the effects of STB on intracellular calcium concentration ([Ca2+]i), another known mediator of intestinal ion and fluid movement. Ca2+ and pH measurements were performed on different cell types including Madin-Darby canine kidney (MDCK), HT-29/C1 intestinal epithelial cells, and primary rat pituitary cells. Ca2+ and pH determinations were performed by simultaneous real-time fluorescence imaging at four emission wavelengths. This allowed dual imaging of the Ca(2+)- and pH-specific ratio dyes (indo-1 and SNARF-1, respectively). STB treatment induced a dose-dependent increase in [Ca2+]i with virtually no effect on internal pH in all of the cell types tested. STB-mediated [Ca2+]i elevation was not inhibited by drugs that block voltage-gated Ca2+ channels including nitrendipine, verapamil (L-type), omega-conotoxin (N-type), and Ni2+ (T-type). The increase in [Ca2+]i was dependent on a source of extracellular Ca2+ and was not affected by prior treatment of MDCK cells with thapsigargin or cyclopiazonic acid, agents that deplete and block internal Ca2+ stores. In contrast to these results, somatostatin and pertussis toxin pretreatment of MDCK cells completely blocked the STB-induced increase in [Ca2+]i. Taken together, these data suggest that STB opens a GTP-binding regulatory protein-linked receptor-operated Ca2+ channel in the plasma membrane. The nature of the STB-sensitive Ca2+ channel is presently under investigation.

Discrimination of duration ratios by pigeons (Columba livia) and humans (Homo sapiens).

Humans (Homo sapiens) were trained on 2 versions of a 2-alternative, forced-choice procedure. First, subjects judged which of 2 successive stimulus durations was longer. Second, subjects judged whether the ratio of the 2 durations was less or greater than a criterion ratio (e.g., 2:1). Accuracy was significantly lower for the task in which the judgment was made according to the ratio of the 2 durations. This result is different than that obtained by Fetterman, Dreyfus, and Stubbs (1989), who trained pigeons (Columbia livia) on a similar pair of tasks and found that pigeons' performance was comparable for the 2 discriminations. Comparisons of the pigeon and human data suggest that humans were more accurate than pigeons when the judgment involved which duration was longer, but that accuracy was comparable for the ratio-based task.

Effect of age on activation of porcine intestinal guanylate cyclase and binding of Escherichia coli heat-stable enterotoxin (STa) to porcine intestinal cells and brush border membranes.

Development of age-dependent resistance to enterotoxigenic Escherichia coli was studied, using isolated enterocytes and brush border membranes (BBM) from 7-day-old and 7-week-old pigs. Binding of 125I-labeled heat-stable (125I-STa) enterotoxin to enterocytes and BBM was specific, temperature- and time-dependent, saturable, and partially reversible. Scatchard analysis revealed a single class of receptors. Mean +/- SD avidity of binding (apparent affinity constant, Ka) of 125I-STa to enterocytes from 7-day-old and 7-week-old pigs was 2.14 +/- 0.29 x 10(8) and 2.72 +/- 0.25 x 10(8) L/mol, respectively. Numbers of STa receptors were calculated to be 64,903 +/- 2,900/enterocyte for 7-day-old pigs and 53,029 +/- 3,117/enterocyte for 7-week-old pigs. Numbers of STa receptors expressed per milligram of BBM protein from 7-day-old pigs were 2.66 x 10(11), compared with 2.29 x 10(11) for BBM from 7-week-old pigs. By 5 minutes after addition of STa to reaction mixtures, intracellular cyclic guanosine monophosphate concentration increased 13.9-fold in enterocytes from 7-day-old pigs and 8.7-fold in enterocytes from 7-week-old pigs. The particulate guanylate cyclase activity associated with BBM from 7-week-old pigs was slightly more sensitive to low amounts of STa, compared with BBM from 7-day-old pigs; however, differences were not observed at intermediate and high amounts. These data indicate that lack of a secretory response to STa by older pigs is not attributable either to decreased numbers of STa receptors or to decreased signal response between the STa receptor and membrane-bound guanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of the STB enterotoxin of Escherichia coli and the role of selected amino acids on its secretion, stability and toxicity.

The methanol-insoluble heat-stable enterotoxin of Escherichia coli (STB) was purified and characterized by automated Edman degradation and tryptic peptide analysis. The amino-terminal residue, Ser-24, confirmed that the first 23 amino acids inferred from the gene sequence were removed during translocation through the E. coli inner membrane. Tryptic peptide analysis coupled with automated Edman degradation revealed that disulphide bonds are formed between residues Cys-33 and Cys-71 and between Cys-44 and Cys-59. Oligonucleotide-directed mutagenesis performed on the STB gene demonstrated that disulphide bond formation does not precede translocation of the polypeptide through the inner membrane and that disulphide bridge formation is a periplasmic event; apparently, elimination of either of two disulphides of STB renders the molecule susceptible to periplasmic proteolysis. In addition, a loop defined by the Cys-44-Cys-59 bond contains at least two amino acids (Arg-52 and Asp-53) required for STB toxic activity.

Monoclonal antibodies specific for the Escherichia coli heat-stable enterotoxin STb.

Protein A-STb and STb-alkaline phosphatase protein fusions were used as immunogen and antigen, respectively, for the generation and screening of monoclonal antibodies to the Escherichia coli heat-stable enterotoxin STb. Following immunization with immunoglobulin G-Sepharose-purified protein A-STb and hybridoma construction, STb-alkaline phosphatase hybrid protein was used in a labeled antigen capture assay to detect the production of STb-specific monoclonal antibody. STb-specific monoclonal antibodies were characterized by using a combination of immunoblotting and synthetic-peptide-based enzyme immunoassay techniques. Four distinct anti-STb antibodies were identified and characterized.

Construction of a bifunctional Escherichia coli heat-stable enterotoxin (STb)-alkaline phosphatase fusion protein.

A fusion between the genes encoding the Escherichia coli STb heat-stable enterotoxin (estB) and alkaline phosphatase (phoA) was constructed, and the expressed protein product was characterized. The STb-alkaline phosphatase protein (STb-PhoA) had an apparent molecular mass of 50,000 daltons and was detected with both monoclonal anti-alkaline phosphatase and polyclonal anti-STb antibodies. Expression of the gene fusion resulted in high-level production of alkaline phosphatase activity, indicating that STb-PhoA was processed and exported into the periplasm of the E. coli host strain. Amino acid sequence analysis of the hybrid protein yielded the sequence Ser-Thr-Gln-Ser-Asn-Lys-Lys, indicating that STb-PhoA was processed during export in a fashion identical to that of native STb (Y. M. Kupersztoch, K. Tachias, C. R. Moomaw, L. A. Dreyfus, R. G. Urban, C. Slaughter, and S. Whipp, J. Bacteriol. 172: 2427-2432, 1990). STb-PhoA was purified from an expressed bacterial lysate by preparative isoelectric focusing. In a rat ligated intestinal loop model, purified STb-PhoA induced highly significant (P less than 0.002) fluid secretion. In addition, the specific activity of STb-PhoA was nearly identical to that of purified STb. Thus, the STb-PhoA hybrid protein represents a readily obtainable source of biologically active (STb) enterotoxin that may prove useful in studies to determine the mode of toxin action.

High-level production of Escherichia coli STb heat-stable enterotoxin and quantification by a direct enzyme-linked immunosorbent assay.

A convenient and sensitive enzyme-linked immunosorbent assay (ELISA) for the STb heat-stable enterotoxin of Escherichia coli was developed and used to quantify STb production by strains with a high level of expression. Based on an antigenic profile of the secreted form of STb, a synthetic peptide (STb3-27) spanning the major predicted epitope was synthesized, coupled to keyhole limpet hemocyanin, and used to immunize rabbits. Anti-STb3-27 antibodies were affinity purified on a synthetic peptide-Sepharose 4B column and used in a direct-binding STb ELISA. Based on a highly purified form of toxin as a standard, the ELISA detected as little as 1 to 2 ng of STb from crude culture filtrates. ELISA data revealed that natural STb-producing strains elaborate little STb in defined-medium cultures relative to that elaborated by a recombinant strain harboring a cloned copy of the estB gene. Replacement of the endogenous STb promoter with any of several highly active promoters, including a bacteriophage T7 promoter, a beta-galactosidase promoter, and a tryptophan-beta-galactosidase hybrid (tac) promoter, increased the yield of STb 10- to 20-fold over levels obtained by an E. coli strain harboring the recombinant estB gene. The high level of STb antigen detected by the ELISA correlated with intestinal secretory activity. The combination of a convenient assay and effective hyperproduction of STb will serve as a basis for a large-scale toxin purification strategy.

Expression and nucleotide sequence analysis of the Legionella pneumophila recA gene.

The nucleotide sequence of the L pneumophila recA gene was determined. The coding region was 1044 nucleotides (348 codons), specifying a 37,934 Da protein. Preceding the recA gene was a tandem set of transcription regulatory sequences and putative LexA binding sites. When expressed in E. coli, the cloned recA gene yield two proteins with molecular weights of approximately 38,000 and 35,500 Da. The larger of these two proteins shared 70.4% and 74.6% identity with the E. coli and Pseudomonas aeruginosa RecA proteins, respectively. The 35,500 Da recA encoded protein was presumed to be the product of translation from Met26 which was preceded by an alternate ribosomal binding site.