Adenylate cyclase toxin from Bordetella pertussis requires posttranslational acylation of lysine 983 for the ability to deliver its catalytic domain to the target cell interior and produce cyclic adenosine monophosphate (cell-invasive activity) and to form transmembrane channels (hemolytic activity). When the toxin is expressed in Escherichia coli, it has reduced hemolytic activity, but comparable cell-invasive activity to that of adenylate cyclase toxin from B. pertussis. In contrast to the native protein from B. pertussis, which is exclusively palmitoylated, recombinant toxin from E. coli is acylated at lysine 983 with about 87% palmitoylated and the remainder myristoylated. Furthermore, the recombinant toxin contains an additional palmitoylation on approximately two-thirds of the lysines at position 860. These observations suggest that the site and nature of posttranslational fatty-acylation can be dictated by the bacterial host used for expression and can have a significant, but selective, effect on protein function.

Adenylate cyclase (AC) toxin from Bordetella pertussis delivers its catalytic domain to the interior of target cells where it converts host ATP to cAMP in a process referred to as intoxication. This toxin also hemolyzes sheep erythrocytes by a mechanism presumed to include pore formation and osmotic lysis. Intoxication and hemolysis appear at strikingly different toxin concentrations and evolve over different time scales, suggesting that different molecular processes may be involved. The present study was designed to test the hypothesis that intoxication and hemolysis occur by distinct mechanisms.Although the hemolytic activity of AC toxin has a lag of >1 h, intoxication starts immediately. Because of this difference, we sought a surrogate or precursor lesion that leads to hemolysis, and potassium efflux has been observed from erythrocytes treated with other poreforming toxins. AC toxin elicits an increase in K ؉ efflux from sheep erythrocytes and Jurkat cells, a human Tcell leukemia line, that begins within minutes of toxin addition. The toxin concentration dependence along with the analysis of the time course suggest that toxin monomers are sufficient to elicit release of K ؉ and to deliver the catalytic domain to the cell interior. Hemolysis, on the other hand, is a highly cooperative event that likely requires a subsequent oligomerization of these individual units. Although induction of K ؉ efflux shares some structural and environmental requirements with both intoxication and hemolysis, it can occur under conditions in which intoxication is reduced or prevented. The data presented here suggest that the transmembrane pathway by which K ؉ is released is separate and distinct from the structure required for intoxication but may be related to, or a precursor of, that which is ultimately responsible for hemolysis.

Acute stress can exert beneficial or detrimental effects on different forms of cognition. In the present study, we assessed the effects of acute restraint stress on different forms of cost/benefit decision-making, and some of the hormonal and neurochemical mechanisms that may underlie these effects. Effort-based decision-making was assessed where rats chose between a low effort/reward (1 press=2 pellets) or high effort/reward option (4 pellets), with the effort requirement increasing over 4 blocks of trials (2, 5, 10, and 20 lever presses). Restraint stress for 1 h decreased preference for the more costly reward and induced longer choice latencies. Control experiments revealed that the effects on decision-making were not mediated by general reductions in motivation or preference for larger rewards. In contrast, acute stress did not affect delay-discounting, when rats chose between a small/immediate vs larger/delayed reward. The effects of stress on decision-making were not mimicked by treatment with physiological doses of corticosterone (1-3 mg/kg). Blockade of dopamine receptors with flupenthixol (0.25 mg/kg) before restraint did not attenuate stress-induced effects on effort-related choice, but abolished effects on choice latencies. These data suggest that acute stress interferes somewhat selectively with cost/benefit evaluations concerning effort costs. These effects do not appear to be mediated solely by enhanced glucocorticoid activity, whereas dopaminergic activation may contribute to increased deliberation times induced by stress. These findings may provide insight into impairments in decision-making and anergia associated with stress-related disorders, such as depression.

SummaryThe cytotoxic effect of adenylate cyclase (AC) toxin from Bordetella pertussis on host cells has been attributed to the production of supraphysiologic levels of cyclic AMP by the toxin. We have tested this hypothesis and show that at least two different mechanisms, cAMP accumulation/ATP depletion and oligomerization/pore formation, contribute, perhaps synergistically, to AC toxin-induced cytotoxicity. Wildtype (WT) AC toxin causes cell death associated with an increase in cAMP, a reduction in ATP, activation of caspases 3/7, and increased annexin V and TUNEL staining. In contrast, a non-acylated, enzymatically active, non-haemolytic form of AC toxin is able to increase cAMP, reduce ATP and elicit annexin V staining, but the decrease in ATP and the annexin staining are transient and there is minimal caspase activation, TUNEL staining and cell death. Mutant AC toxins defective in either enzymatic activity or the ability to deliver their enzymatic domain are able to kill J774 cells, without cAMP production, and with minimal caspase activation and TUNEL staining. Comparison of the potencies of WT toxin and those of mutants that only increase cAMP or only create transmembrane pores establishes that at least two mechanisms are contributory and that simply the production of cAMP is not enough to account for the cytotoxicity produced by AC toxin.

In these studies, the Bordetella pertussis adenylate cyclase toxin-hemolysin homology to the Escherichia coli hemolysin is extended with the finding of cyaC, a homolog to the E. coli hlyC gene, which is required for the production of a functional hemolysin molecule in E. coli. Mutations produced in the chromosome of B. pertussis upstream from the structural gene for the adenylate cyclase toxin revealed a region which was necessary for toxin and hemolytic activities of the molecule. These mutants produced the 216-kDa adenylate cyclase toxin as determined by Western blot (immunoblot) analysis. The adenylate cyclase enzymatic activities of these mutants were equivalent to that of wild type, but toxin activities were <1% of that of wild type, and the mutants were nonhemolytic on blood agar plates and in in vitro assays. The upstream region restored hemolytic activity when returned in trans to the mutant strains. This genetic complementation defined a gene which acts in trans to activate the adenylate cyclase toxin posttranslationally. Sequence analysis of the upstream region defined an open reading frame with homology to the E. coli hlyC gene. In contrast to E. coli, this open reading frame is oriented oppositely from the adenylate cyclase toxin structural gene.